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This invention was made with government support under Grant Nos.
AI048694, GM50694, GM65307 and GM073216 awarded by the National
Institutes of Health. The government has certain rights in this
invention.

Parent Case Text

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a National Stage Application filed under 35 U.S.C.
.sctn.371 and claims priority to International Application No.
PCT/US07/011466, filed May 11, 2007, which application claims priority
under 35 U.S.C. .sctn.119 to U.S. Provisional Application Ser. No.
60/800,564 filed May 12, 2006, the disclosures of which are incorporated
herein by reference. Related application PCT/US06/14486 is incorporated
herein by reference.

Claims

The invention claimed is:

1. A method for treating a bacterial infection, comprising: (a) providing a composition comprising a compound having a Formula I or a Formula II: ##STR00016## wherein:
m is 0, 1, 2 or 3; n is an integer between 1 and 10 inclusive; each D and E are each independently selected from the group consisting of: H, aryl, substituted aryl, alkyl, substituted alkyl, carboxyl, aminocarbonyl, alkylsulfonylaminocarboxyl,
alkoxycarbonyl, and halo; M.sup.1 M.sup.2, and M.sup.3 are each independently selected from the group consisting of metals, ammonium and esters thereof; R.sup.1 is selected from the group consisting of: H, aryl, substituted aryl, alkyl, substituted
alkyl, carboxyl, aminocarbonyl, alkylsulfonylaminocarboxyl, alkoxycarbonyl, and halo, or R.sup.1 and R.sup.2, together with the carbons to which they are bound, can be joined to form a 4 to 7 membered ring or a substituted 4 to 7 membered ring; R.sup.2
is selected from the group consisting of: H, aryl, substituted aryl, alkyl, substituted alkyl, carboxyl, aminocarbonyl, alkylsulfonylaminocarboxyl, alkoxycarbonyl, and halo, or R.sup.2 and R.sup.1, together with the carbons to which they are bound, can
be joined to form a 4 to 7 membered ring or a substituted 4 to 7 membered ring, or R.sup.2 and R.sup.3, together with the carbons to which they are bound, can be joined to form a 4 to 7 membered ring or a substituted 4 to 7 membered ring; R.sup.3 is
selected from the group consisting of: H, aryl, substituted aryl, alkyl, substituted alkyl, carboxyl, aminocarbonyl, alkylsulfonylaminocarboxyl, alkoxycarbonyl, and halo, or R.sup.3 and R.sup.2, together with the carbons to which they are bound, can be
joined to form a 4 to 7 membered ring or a substituted 4 to 7 membered ring, or R.sup.3 and R.sup.4, together with the carbons to which they are bound, can be joined to form a 4 to 7 membered ring or a substituted 4 to 7 membered ring; R.sup.4 is
selected from the group consisting of: H, aryl, substituted aryl, alkyl, substituted alkyl, carboxyl, aminocarbonyl, alkylsulfonylaminocarboxyl, alkoxycarbonyl, and halo, or R.sup.4 and R.sup.3, together with the carbons to which they are bound, can be
joined to form a 4 to 7 membered ring or a substituted 4 to 7 membered ring; R.sup.5, R.sup.6, R.sup.7, R.sup.8, and R.sup.9 are each independently selected from the group consisting of: H, aryl, substituted aryl, alkyl, substituted alkyl, carboxyl,
aminocarbonyl, alkylsulfonylaminocarboxyl, alkoxycarbonyl, and halo; L.sup.1 is --S--, --SO--, --SO.sub.2--, --O--, --N(R.sup.19)--, or --C(R.sup.20)(R.sup.21)--; wherein R.sup.19, R.sup.20 and R.sup.21 are each independently selected from the group
consisting of: H, aryl, substituted aryl, alkyl, substituted alkyl, carboxyl, aminocarbonyl, alkylsulfonylaminocarboxyl, alkoxycarbonyl, and halo; ##STR00017## wherein: x is 0, 1 , 2, or 3; y is an integer between 1 and 10 inclusive; each G and J are
independently selected from the group consisting of: H, aryl, substituted aryl, alkyl, substituted alkyl, carboxyl, aminocarbonyl, alkylsulfonylaminocarboxyl, alkoxycarbonyl, and halo; M.sup.4, M.sup.5, and M.sup.6 are each independently selected from
the group consisting of metals, ammonium and esters thereof; R.sup.10 is selected from the group consisting of: H, aryl, substituted aryl, alkyl, substituted alkyl, carboxyl, aminocarbonyl, alkylsulfonylaminocarboxyl, alkoxycarbonyl, and halo, or
R.sup.10 and R.sup.11, together with the carbons to which they are bound, can be joined to form a 4 to 7 membered ring or a substituted 4 to 7 membered ring; R.sup.11 is selected from the group consisting of: H, aryl, substituted aryl, alkyl,
substituted alkyl, carboxyl, aminocarbonyl, alkylsulfonylaminocarboxyl, alkoxycarbonyl, and halo, or R.sup.11 and R.sup.10, together with the carbons to which they are bound, can be joined to form a 4 to 7 membered ring or a substituted 4 to 7 membered
ring, or R.sup.11 and R.sup.12, together with the carbons to which they are bound, can be joined to form a 4 to 7 membered ring or a substituted 4 to 7 membered ring; R.sup.12 is selected from the group consisting of: H, aryl, substituted aryl, alkyl,
substituted alkyl, carboxyl, aminocarbonyl, alkylsulfonylaminocarboxyl, alkoxycarbonyl, and halo, or R.sup.12 and R.sup.11, together with the carbons to which they are bound, can be joined to form a 4 to 7 membered ring or a substituted 4 to 7 membered
ring, or R.sup.12 and R.sup.13, together with the carbons to which they are bound, can be joined to form a 4 to 7 membered ring or a substituted 4 to 7 membered ring; R.sup.13 is selected from the group consisting of: H, aryl, substituted aryl, alkyl,
substituted alkyl, carboxyl, aminocarbonyl, alkylsulfonylaminocarboxyl, alkoxycarbonyl, and halo, or R.sup.13 and R.sup.12, together with the carbons to which they are bound, can be joined to form a 4 to 7 membered ring or a substituted 4 to 7 membered
ring; R.sup.14, R.sup.15, R.sup.16, R.sup.17, and R.sup.18 are each independently selected from the group consisting of: H, aryl, substituted aryl, alkyl, substituted alkyl, carboxyl, aminocarbonyl, alkylsulfonylaminocarboxyl, alkoxycarbonyl, and halo;
L.sup.2 is --S--, --SO--, --SO.sub.2--, --O--, --N(R.sup.22)--, or --C(R.sup.23)(R.sup.24)--; wherein R.sup.22, R.sup.23 and R.sup.24 are each independently selected from the group consisting of: H, aryl, substituted aryl, alkyl, substituted alkyl,
carboxyl, aminocarbonyl, alkylsulfonylaminocarboxyl, alkoxycarbonyl, and halo; and (b) contacting the composition with the bacterial microbe, thereby treating a bacterial infection, wherein the bacterial microbe expresses a carotenoid and wherein the
compound inhibits the production of the carotenoid rendering the bacterial microbe more susceptible to oxidative damage.

2. The method of claim 1, wherein: m is zero or one; n is an integer between 1 and 5, inclusive; each D and E are independently selected from H, alkyl, substituted alkyl and halo; R.sup.1 is selected from the group consisting of: H, aryl,
substituted aryl, alkyl, substituted alkyl, and halo or R.sup.1 and R.sup.2, together with the carbons to which they are bound, can be joined to form a furan, substituted furan, pyran, substituted pyran, pyrrole, substituted pyrrole, pyridine,
substituted pyridine, aromatic, or substituted aromatic, cyclopentyl, substituted cyclopentyl, cyclohexyl, or substituted cyclohexyl ring; R.sup.2 is selected from the group consisting of: H, aryl, substituted aryl, alkyl, substituted alkyl, and halo or
R.sup.2 and R.sup.1, together with the carbons to which they are bound, can be joined to form a furan, substituted furan, pyran, substituted pyran, pyrrole, substituted pyrrole, pyridine, substituted pyridine, aromatic, or substituted aromatic,
cyclopentyl, substituted cyclopentyl, cyclohexyl, or substituted cyclohexyl ring, or R.sup.2 and R.sup.3, together with the carbons to which they are bound, can be joined to form a furan, substituted furan, pyran, substituted pyran, pyrrole, substituted
pyrrole, pyridine, substituted pyridine, aromatic, or substituted aromatic, cyclopentyl, substituted cyclopentyl, cyclohexyl, or substituted cyclohexyl ring; R.sup.3 is selected from the group consisting of: H, aryl, substituted aryl, alkyl, substituted
alkyl, and halo or R.sup.3 and R.sup.2, together with the carbons to which they are bound, can be joined to form a furan, substituted furan, pyran, substituted pyran, pyrrole, substituted pyrrole, pyridine, substituted pyridine, aromatic, or substituted
aromatic, cyclopentyl, substituted cyclopentyl, cyclohexyl, or substituted cyclohexyl ring, or R.sup.3 and R.sup.4, together with the carbons to which they are bound, can be joined to form a furan, substituted furan, pyran, substituted pyran, pyrrole,
substituted pyrrole, pyridine, substituted pyridine, aromatic, or substituted aromatic, cyclopentyl, substituted cyclopentyl, cyclohexyl, or substituted cyclohexyl ring; R.sup.4 is selected from the group consisting of: H, aryl, substituted aryl, alkyl,
substituted alkyl, and halo or R.sup.4 and R.sup.3, together with the carbons to which they are bound, can be joined to form a furan, substituted furan, pyran, substituted pyran, pyrrole, substituted pyrrole, pyridine, substituted pyridine, aromatic, or
substituted aromatic, cyclopentyl, substituted cyclopentyl, cyclohexyl, or substituted cyclohexyl ring; R.sup.5, R.sup.6, R.sup.7, R.sup.8, and R.sup.9 are each independently selected from the group consisting of: H, aryl, substituted aryl, alkyl,
substituted alkyl, and halo; R.sup.19, R.sup.20 and R.sup.21 are each independently selected from the group consisting of: H, alkyl, and substituted alkyl; M.sup.1, M.sup.2, and M.sup.3 are each independently alkali metals; x is zero or one; y is an
integer between 1 and 5, inclusive; each G and J are independently selected from H, alkyl, substituted alkyl and halo; R.sup.10 is selected from the group consisting of: H, aryl, substituted aryl, alkyl, substituted alkyl, and halo or R.sup.10 and
R.sup.11, together with the carbons to which they are bound, can be joined to form a furan, substituted furan, pyran, substituted pyran, pyrrole, substituted pyrrole, pyridine, substituted pyridine, aromatic, or substituted aromatic, cyclopentyl,
substituted cyclopentyl, cyclohexyl, or substituted cyclohexyl ring; R.sup.11 is selected from the group consisting of: H, aryl, substituted aryl, alkyl, substituted alkyl, and halo or R.sup.11 and R.sup.10, together with the carbons to which they are
bound, can be joined to form a furan, substituted furan, pyran, substituted pyran, pyrrole, substituted pyrrole, pyridine, substituted pyridine, aromatic, or substituted aromatic, cyclopentyl, substituted cyclopentyl, cyclohexyl, or substituted
cyclohexyl ring, or R.sup.11 and R.sup.12, together with the carbons to which they are bound, can be joined to form a furan, substituted furan, pyran, substituted pyran, pyrrole, substituted pyrrole, pyridine, substituted pyridine, aromatic, or
substituted aromatic, cyclopentyl, substituted cyclopentyl, cyclohexyl, or substituted cyclohexyl ring; R.sup.12 is selected from the group consisting of: H, aryl, substituted aryl, alkyl, substituted alkyl, and halo or R.sup.12 and R.sup.11, together
with the carbons to which they are bound, can be joined to form a furan, substituted furan, pyran, substituted pyran, pyrrole, substituted pyrrole, pyridine, substituted pyridine, aromatic, or substituted aromatic, cyclopentyl, substituted cyclopentyl,
cyclohexyl, or substituted cyclohexyl ring, or R.sup.12 and R.sup.13, together with the carbons to which they are bound, can be joined to form a furan, substituted furan, pyran, substituted pyran, pyrrole, substituted pyrrole, pyridine, substituted
pyridine, aromatic, or substituted aromatic, cyclopentyl, substituted cyclopentyl, cyclohexyl, or substituted cyclohexyl ring; R.sup.13 is selected from the group consisting of: H, aryl, substituted aryl, alkyl, substituted alkyl, and halo or R.sup.13
and R.sup.12, together with the carbons to which they are bound, can be joined to form a furan, substituted furan, pyran, substituted pyran, pyrrole, substituted pyrrole, pyridine, substituted pyridine, aromatic, or substituted aromatic, cyclopentyl,
substituted cyclopentyl, cyclohexyl, or substituted cyclohexyl ring; R.sup.14, R.sup.15, R.sup.16, R.sup.17, and R.sup.18 are each independently selected from the group consisting of: H, aryl, substituted aryl, alkyl, substituted alkyl, and halo;
R.sup.22, R.sup.23, and R.sup.24 are each independently selected from the group consisting of: H, alkyl, and substituted alkyl; M.sup.4, M.sup.5, and M.sup.6 are each independently alkali metals.

3. The method of claim 1, wherein: m is one; n is three; each D and E are H; R.sup.1 is H, F, or benzyl; R.sup.2 is H, F or CF.sub.3; R.sup.3 is H, F, Cl, CF.sub.3, t-butyl, n-propyl, or benzyl; R.sup.4 is H or F; R.sup.5, R.sup.6,
R.sup.7 and R.sup.9 are each H; R.sup.8 is H or F; M.sup.1, M.sup.2, and M.sup.3 are each potassium; L.sup.1 is --O--, --NH--or CH.sub.2; x is zero or one; y is three; each G and J are H; R.sup.10, R.sup.11, R.sup.13, R.sup.14, R.sup.15, R.sup.16,
R.sup.17, and R.sup.18 are each H; R.sup.12 is H or CH.sub.3 M.sup.4, M.sup.5, and M.sup.6 are each potassium; L.sup.2 is --O--.

4. The method of claim 1, wherein the compound is selected from the group consisting of: ##STR00018## ##STR00019##

7. The method of claim 1, wherein the compound is of Formula III ##STR00020##

8. The method of claim 1, wherein the compound further comprises a pharmaceutically acceptable carrier.

9. The method of claim 1, wherein the compound is formulated or manufactured as a tablet, a pill, a capsule, a coating, a microsphere, liposome, a wipe, a lotion, a cream, a gel, an ointment or a spray; or formulated for delivery
intramuscularly, intravenously, intraperitoneally, subcutaneously, by inhalation or transdermally.

10. The method of claim 1, wherein the bacterial infection is a Staphylococcus infection, or a bacterial infection caused by a Staphylococcus sp. or a Staphylococcus aureus.

11. The method of claim 1, wherein the contacting of the bacterial microbe with the compound is in vivo or in vitro.

12. A method of inhibiting the growth of a bacterial microbe, comprising (a) contacting the bacterial microbe in vivo or in vitro with a compound, wherein the compound has a Formula I or a Formula II: ##STR00021## wherein: m is 0, 1, 2 or 3; n
is an integer between 1 and 10 inclusive; each D and E are each independently selected from the group consisting of: H, aryl, substituted aryl, alkyl, substituted alkyl, carboxyl, aminocarbonyl, alkylsulfonylaminocarboxyl, alkoxycarbonyl, and halo;
M.sup.1 M.sup.2, and M.sup.3 are each independently selected from the group consisting of metals, ammonium and esters thereof; R.sup.1 is selected from the group consisting of: H, aryl, substituted aryl, alkyl, substituted alkyl, carboxyl,
aminocarbonyl, alkylsulfonylaminocarboxyl, alkoxycarbonyl, and halo, or R.sup.1 and R.sup.2, together with the carbons to which they are bound, can be joined to form a 4 to 7 membered ring or a substituted 4 to 7 membered ring; R.sup.2 is selected from
the group consisting of: H, aryl, substituted aryl, alkyl, substituted alkyl, carboxyl, aminocarbonyl, alkylsulfonylaminocarboxyl, alkoxycarbonyl, and halo, or R.sup.2 and R.sup.1, together with the carbons to which they are bound, can be joined to form
a 4 to 7 membered ring or a substituted 4 to 7 membered ring, or R.sup.2 and R.sup.3, together with the carbons to which they are bound, can be joined to form a 4 to 7 membered ring or a substituted 4 to 7 membered ring; R.sup.3 is selected from the
group consisting of: H, aryl, substituted aryl, alkyl, substituted alkyl, carboxyl, aminocarbonyl, alkylsulfonylaminocarboxyl, alkoxycarbonyl, and halo, or R.sup.3 and R.sup.2, together with the carbons to which they are bound, can be joined to form a 4
to 7 membered ring or a substituted 4 to 7 membered ring, or R.sup.3 and R.sup.4, together with the carbons to which they are bound, can be joined to form a 4 to 7 membered ring or a substituted 4 to 7 membered ring; R.sup.4 is selected from the group
consisting of: H, aryl, substituted aryl, alkyl, substituted alkyl, carboxyl, aminocarbonyl, alkylsulfonylaminocarboxyl, alkoxycarbonyl, and halo, or R.sup.4 and R.sup.3, together with the carbons to which they are bound, can be joined to form a 4 to 7
membered ring or a substituted 4 to 7 membered ring; R.sup.5, R.sup.6, R.sup.7, R.sup.8, and R.sup.9 are each independently selected from the group consisting of: H, aryl, substituted aryl, alkyl, substituted alkyl, carboxyl, aminocarbonyl,
alkylsulfonylaminocarboxyl, alkoxycarbonyl, and halo; L.sup.1 is --S--, --SO--, --SO.sub.2--, --O--, --N(R.sup.19)--, or --C(R.sup.20)(R.sup.21)--; wherein R.sup.19, R.sup.20 and R.sup.21 are each independently selected from the group consisting of: H,
aryl, substituted aryl, alkyl, substituted alkyl, carboxyl, aminocarbonyl, alkylsulfonylaminocarboxyl, alkoxycarbonyl, and halo; ##STR00022## wherein: x is 0 , 1 , 2 , or 3; y is an integer between 1 and 10 inclusive; each G and J are independently
selected from the group consisting of: H, aryl, substituted aryl, alkyl, substituted alkyl, carboxyl, aminocarbonyl, alkylsulfonylaminocarboxyl, alkoxycarbonyl, and halo; M.sup.4, M.sup.5, and M.sup.6 are each independently selected from the group
consisting of metals, ammonium and esters thereof; R.sup.10 is selected from the group consisting of: H, aryl, substituted aryl, alkyl, substituted alkyl, carboxyl, aminocarbonyl, alkylsulfonylaminocarboxyl, alkoxycarbonyl, and halo, or R.sup.10 and
R.sup.11, together with the carbons to which they are bound, can be joined to form a 4 to 7 membered ring or a substituted 4 to 7 membered ring; R.sup.11 is selected from the group consisting of: H, aryl, substituted aryl, alkyl, substituted alkyl,
carboxyl, aminocarbonyl, alkylsulfonylaminocarboxyl, alkoxycarbonyl, and halo, or R.sup.11 and R.sup.10, together with the carbons to which they are bound, can be joined to form a 4 to 7 membered ring or a substituted 4 to 7 membered ring, or R.sup.11
and R.sup.12, together with the carbons to which they are bound, can be joined to form a 4 to 7 membered ring or a substituted 4 to 7 membered ring; R.sup.12 is selected from the group consisting of: H, aryl, substituted aryl, alkyl, substituted alkyl,
carboxyl, aminocarbonyl, alkylsulfonylaminocarboxyl, alkoxycarbonyl, and halo, or R.sup.12 and R.sup.11, together with the carbons to which they are bound, can be joined to form a 4 to 7 membered ring or a substituted 4 to 7 membered ring, or R.sup.12
and R.sup.13, together with the carbons to which they are bound, can be joined to form a 4 to 7 membered ring or a substituted 4 to 7 membered ring; R.sup.13 is selected from the group consisting of: H, aryl, substituted aryl, alkyl, substituted alkyl,
carboxyl, aminocarbonyl, alkylsulfonylaminocarboxyl, alkoxycarbonyl, and halo, or R.sup.13 and R.sup.12, together with the carbons to which they are bound, can be joined to form a 4 to 7 membered ring or a substituted 4 to 7 membered ring; R.sup.14,
R.sup.15, R.sup.16, R.sup.17, and R.sup.18 are each independently selected from the group consisting of: H, aryl, substituted aryl, alkyl, substituted alkyl, carboxyl, aminocarbonyl, alkylsulfonylaminocarboxyl, alkoxycarbonyl, and halo; L.sup.2 is
--S--, --SO--, --SO.sub.2--, --O--, --N(R.sup.22)--, or --C(R.sup.23)(R.sup.24)--; wherein R.sup.22, R.sup.23 and R.sup.24 are each independently selected from the group consisting of: H, aryl, substituted aryl, alkyl, substituted alkyl, carboxyl,
aminocarbonyl, alkylsulfonylaminocarboxyl, alkoxycarbonyl, and halo.

13. The method of claim 12, wherein the contacting in vivo is to treat a Staphylococcus infection, or Staphylococcus aureus infection.

14. The method of claim 13, wherein (a) the contacting is on a surface of a bacterial microbe comprising a carotenoid (b) the bacteria is gram positive, (c) bacteria is Staphylococcus aureus or S. epidermidis; (e) the bacteria is gram
negative, (e) the bacteria is selected from the group consisting of E. coli, P. aeruginosa, and S. typhimurium.

Description

BACKGROUND

Ogston (1881) coined the genus Staphylococcus to describe grapelike clusters of bacteria (staphylo=grape, Gr.) recovered in pus from surgical abscesses. Shortly thereafter, Rosenbach (1884) isolated this pathogen in pure culture, and proposed
the species name S. aureus (golden, Lat.) for its characteristic surface pigmentation in comparison to less virulent staphylococci that normally colonize the skin surface.

Entering its seventh decade, the era of antimicrobial therapy has greatly reduced morbidity and mortality from infectious diseases. However, the emergence of resistant microorganisms has now reached epidemic proportions and poses great
challenges to the medical community. Worrisome trends are particularly evident in the pre-eminent Gram-positive bacterial pathogen S. aureus, which has become increasingly unresponsive to first-line antibiotic therapies. S. aureus is probably the most
common cause of life-threatening acute bacterial infections in the world, and is capable of causing a diverse array of diseases, ranging in severity from a simple boil or impetigo to fulminant sepsis or toxic shock syndrome. S. aureus is the single
leading cause of bacteremia, hospital-related (nosocomial) infections, skin and soft tissue infections, wound infections, and bone and joint infections. It is one of the most common agents of endocarditis and food poisoning.

National prospective surveillance of over 24,000 invasive bacterial isolates show disease-associated S. aureus strains with methicillin resistance (MRSA) have increased from 22% in 1995 to 57% currently. MRSA are now frequently identified in
community-acquired infections as well as in hospital settings. An urgent need exists for discovery of novel classes of antibiotics to address this genuine public health crisis. A half-century of synthesizing analogs based on <10 antibacterial
scaffolds has resulted in the development and marketing of >100 antibacterial agents but, with the exception of the oxazolidinone core, no new scaffolds have emerged in the past 30 years to address the emerging resistance problems.

Classic antibiotic approaches attempt to kill or suppress growth of bacteria by targeting essential cell functions such as cell wall biosynthesis, protein synthesis, DNA replication, RNA polymerase, or metabolic pathways. These conventional
therapies run a higher risk of toxicity since many of these cell functions are also essential to mammalian cells and require fine molecular distinction between the microbial target and host cell counterpart(s). Second, the repetitive use of the same
essential targets means that when a bacterium evolves resistance to a particular antibiotic agent during therapy, it can become simultaneously cross resistant to other agents acting on the same target, even though the bacterium has never been exposed to
the other agents. Third, conventional therapies exert a "life-or-death" challenge upon the bacterium, and thus a strong selective pressure to evolve resistance to the antimicrobial agent. Finally, many current antibiotics have very broad spectrums of
activity, with the side effect of eradicating many components of the normal flora, leading to undesired complications such as Clostridium difficile colitis or secondary fungal infections (e.g. Candida).

The emergence of MRSA has compromised the clinical utility of methicillin and related antibiotics (oxacillin, dicloxacillin) and all cephalosporings (e.g. cefazolin, cephalexin) in empiric therapy of S. aureus infections. MRSA often have
significant levels of resistance to macrolides (e.g. erythromycin), beta-lactamase inhibitor combinations (e.g. Unasyn, Augmentin) and fluoroquinolones (e.g. ciprofloxacin), and are occasionally resistant to clindamycin, trimethoprim/sulfamethoxisol
(Bactrim), and rifampin. In serious S. aureus infection, intravenous Vancomycin is the last resort, but there have now also been alarming reports of S. aureus resistance to vancomycin, an intravenous antibiotic commonly used to treat MRSA.

New anti-MRSA agents such as linezolid (Zyvox or quinupristin/dalfopristin (Synercid), both of which utilize the traditional target of binding to the ribosomal subunits to inhibit RNA synthesis, are quite expensive.

SUMMARY

The invention demonstrates that carotenoids serve as virulence/resistance factors in microbes. In one specific example, the invention demonstrates that S. aureus carotenoid is a virulence factor that impairs neutrophil killing and promotes
disease pathogenesis by virtue of its antioxidant properties, 2) evidence that pharmacological inhibition of the carotenoid pigment production renders the organism more susceptible to oxidants and blood killing; and (3) evidence that squalene synthesis
inhibitors can block Staphylococcus aureus pigment production, and that squalene synthesis inhibition can reduce Staphylococcus aureus pathogenicity in vivo.

The invention provides a compound comprising A compound represented by formula I or II:

##STR00001## wherein: m is 0, 1, 2 or 3; n is an integer between 1 and 10 inclusive; each D and E are each independently selected from the group consisting of: H, aryl, substituted aryl, alkyl, substituted alkyl, carboxyl, aminocarbonyl,
alkylsulfonylaminocarboxyl, alkoxycarbonyl, and halo; M.sup.1, M.sup.2, and M.sup.3 are each independently selected from the group consisting of metals, ammonium and esters thereof; R.sup.1 is selected from the group consisting of: H, aryl, substituted
aryl, alkyl, substituted alkyl, carboxyl, aminocarbonyl, alkylsulfonylaminocarboxyl, alkoxycarbonyl, and halo, or R.sup.1 and R.sup.2, together with the carbons to which they are bound, can be joined to form a 4 to 7 membered ring or a substituted 4 to 7
membered ring; R.sup.2 is selected from the group consisting of: H, aryl, substituted aryl, alkyl, substituted alkyl, carboxyl, aminocarbonyl, alkylsulfonylaminocarboxyl, alkoxycarbonyl, and halo, or R.sup.2 and R.sup.1, together with the carbons to
which they are bound, can be joined to form a 4 to 7 membered ring or a substituted 4 to 7 membered ring, or R.sup.2 and R.sup.3, together with the carbons to which they are bound, can be joined to form a 4 to 7 membered ring or a substituted 4 to 7
membered ring; R.sup.3 is selected from the group consisting of: H, aryl, substituted aryl, alkyl, substituted alkyl, carboxyl, aminocarbonyl, alkylsulfonylaminocarboxyl, alkoxycarbonyl, and halo, or R.sup.3 and R.sup.2, together with the carbons to
which they are bound, can be joined to form a 4 to 7 membered ring or a substituted 4 to 7 membered ring, or R.sup.3 and R.sup.4, together with the carbons to which they are bound, can be joined to form a 4 to 7 membered ring or a substituted 4 to 7
membered ring; R.sup.4 is selected from the group consisting of: H, aryl, substituted aryl, alkyl, substituted alkyl, carboxyl, aminocarbonyl, alkylsulfonylaminocarboxyl, alkoxycarbonyl, and halo, or R.sup.4 and R.sup.3, together with the carbons to
which they are bound, can be joined to form a 4 to 7 membered ring or a substituted 4 to 7 membered ring; R.sup.5, R.sup.6, R.sup.7, R.sup.8, and R.sup.9 are each independently selected from the group consisting of: H, aryl, substituted aryl, alkyl,
substituted alkyl, carboxyl, aminocarbonyl, alkylsulfonylaminocarboxyl, alkoxycarbonyl, and halo; L.sup.1 is --S--, --SO--, --SO.sub.2--, --O--, --N(R.sup.19)--, or --C(R.sup.20)(R.sup.21)--; wherein R.sup.19, R.sup.20 and R.sup.21 are each independently
selected from the group consisting of: H, aryl, substituted aryl, alkyl, substituted alkyl, carboxyl, aminocarbonyl, alkylsulfonylaminocarboxyl, alkoxycarbonyl, and halo;

##STR00002## wherein: x is 0, 1, 2, or 3; y is an integer between 1 and 10 inclusive; each G and J are independently selected from the group consisting of: H, aryl, substituted aryl, alkyl, substituted alkyl, carboxyl, aminocarbonyl,
alkylsulfonylaminocarboxyl, alkoxycarbonyl, and halo; M.sup.4, M.sup.5, and M.sup.6 are each independently selected from the group consisting of metals, ammonium and esters thereof; R.sup.10 is selected from the group consisting of: H, aryl, substituted
aryl, alkyl, substituted alkyl, carboxyl, aminocarbonyl, alkylsulfonylaminocarboxyl, alkoxycarbonyl, and halo, or R.sup.10 and R.sup.11, together with the carbons to which they are bound, can be joined to form a 4 to 7 membered ring or a substituted 4 to
7 membered ring; R.sup.11 is selected from the group consisting of: H, aryl, substituted aryl, alkyl, substituted alkyl, carboxyl, aminocarbonyl, alkylsulfonylaminocarboxyl, alkoxycarbonyl, and halo, or R.sup.11 and R.sup.10, together with the carbons to
which they are bound, can be joined to form a 4 to 7 membered ring or a substituted 4 to 7 membered ring, or R.sup.11 and R.sup.12, together with the carbons to which they are bound, can be joined to form a 4 to 7 membered ring or a substituted 4 to 7
membered ring; R.sup.12 is selected from the group consisting of: H, aryl, substituted aryl, alkyl, substituted alkyl, carboxyl, aminocarbonyl, alkylsulfonylaminocarboxyl, alkoxycarbonyl, and halo, or R.sup.12 and R.sup.11, together with the carbons to
which they are bound, can be joined to form a 4 to 7 membered ring or a substituted 4 to 7 membered ring, or R.sup.12 and R.sup.13, together with the carbons to which they are bound, can be joined to form a 4 to 7 membered ring or a substituted 4 to 7
membered ring; R.sup.13 is selected from the group consisting of: H, aryl, substituted aryl, alkyl, substituted alkyl, carboxyl, aminocarbonyl, alkylsulfonylaminocarboxyl, alkoxycarbonyl, and halo, or R.sup.13 and R.sup.12, together with the carbons to
which they are bound, can be joined to form a 4 to 7 membered ring or a substituted 4 to 7 membered ring; R.sup.14, R.sup.15, R.sup.16, R.sup.17 and R.sup.18 are each independently selected from the group consisting of: H, aryl, substituted aryl, alkyl,
substituted alkyl, carboxyl, aminocarbonyl, alkylsulfonylaminocarboxyl, alkoxycarbonyl, and halo; L.sup.2 is --S--, --SO--, --SO.sub.2--, --O--, --N(R.sup.22)--, or --C(.sup.23)(R.sup.24)--; wherein R.sup.22, R.sup.23 and R.sup.24 are each independently
selected from the group consisting of: H, aryl, substituted aryl, alkyl, substituted alkyl, carboxyl, aminocarbonyl, alkylsulfonylaminocarboxyl, alkoxycarbonyl, and halo.

In another aspect, m is zero or one; n is and integer between 1 and .sup.5, inclusive; each D and E are independently selected from H, alkyl, substituted alkyl and halo; R.sup.1, R.sup.2, R.sup.3, R.sup.4, R.sup.5, R.sup.6, R.sup.7, R.sup.8, and
R.sup.9 are each independently selected from the group consisting of: H, aryl, substituted aryl, alkyl, substituted alkyl, and halo; R.sup.19, R.sup.20 and R.sup.21 are each independently selected from the group consisting of: H, alkyl, and substituted
alkyl; M.sup.1, M.sup.2, and M.sup.3 are each independently alkali metals; x is zero or one; y is and integer between 1 and 5, inclusive; each G and J are independently selected from H, alkyl, substituted alkyl and halo; R.sup.10, R.sup.11, R.sup.12,
R.sup.13, R.sup.14, R.sup.15, R.sup.16, R.sup.17, and R.sup.18 are each independently selected from the group consisting of: H, aryl, substituted aryl, alkyl, substituted alkyl, and halo; R.sup.22, R.sup.23 and R.sup.24 are each independently selected
from the group consisting of: H, alkyl, and substituted alkyl; M.sup.4, M.sup.5, and M.sup.6 are each independently alkali metals. In yet another aspect, m is one; n is three; each D and E are H; R.sup.1 is H, F, or benzyl; R.sup.2 is H, F or CF.sub.3;
R.sup.3 is H, F, CL, CF.sub.3, t-butyl, n-propyl, or benzyl; R.sup.4is H or F; R.sup.5, R.sup.6, R.sup.7 and R.sup.9 are each H; R.sup.8 is H or F; M.sup.1, M.sup.2, and M.sup.3 are each potassium; L.sup.1 is --O--, --NH-- or CH.sub.2; x is zero or one;
y is three; each G and J are H; R.sup.10, R.sup.11, R.sup.12, R.sup.13, R.sup.14, R.sup.15, R.sup.16, R.sup.17, and R.sup.18 are each H; R.sup.12 is H or CH.sub.3, M.sup.4, M.sup.5, and M.sup.6 are each potassium; L.sup.2 is --O--.

The invention also provides compounds comprising the following structures. Such compounds are useful inhibitors of dehydrosqualene synthase activity.

##STR00003## ##STR00004## Pharmaceutically acceptable salts, prodrugs and enantiomers of any of the above are also encompassed by the invention.

The invention also provides a method of preventing or treating a bacterial infection, comprising administering to a subject inflicted with the infection an agent that inhibits the production and/or activity of a carotenoid in the bacteria. In
one embodiment, the bacterial infection is a Staphylococcus infection. In another embodiment, the bacteria is a Staphylococcus sp. In yet a further embodiment, the bacteria is Staphylococcus aureus.

The invention also provides a method of preventing or treating a bacterial infection comprising administering to a subject inflicted with the infection the compound of Formula I or those, wherein the compound of Formulas I and II inhibit
squalene synthase.

The invention also provides a method of preventing, treating or improving the effective treatment of MRSA by contacting a microbe expressing a squalene synthase with a squalene synthase inhibitor.

FIG. 4A-B shows the inhibition of Staphylococcus aureus pigment production increases oxidant sensitivity and phagocytic clearance. Wild-type and .DELTA.CrtM mutant S. aureus were cultured in the presence or absence of SKF 525-A at the indicated
concentrations. Depicted are the observed effects on A) pigmentation phenotype, B) singlet oxygen susceptibility, and C) survival in murine whole blood. Results shown are representative of at least three experiments.

FIG. 8 shows the effect of the compound of Formula III at 40 mM on whole blood killing of S. aureus. There was a major enhancement in whole blood killing of S. aureus with a .sup..about.3 fold enhancement at 40.about.3 fold enhancement at 40
.mu.M (ED.sub.50 .about.20 .mu.M).

FIG. 9A-B shows that in the presence of the compound of Formula III in an in vivo mouse model of S. aureus skin infection there was a major decrease in lesion size at (A) 200 .mu.M or (B) 1000 .mu.M with no apparent ill effects.

FIG. 10 shows an additional study using a systemic (intraperitoneal) challenge model in mice that pigment conferred a survival advantage to the bacteria. Here cfu counts are measured in the kidneys.

As used herein and in the appended claims, the singular forms "a," "and," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a compound" includes a plurality of such compounds and
reference to "the cell" includes reference to one or more cells known to those skilled in the art, and so forth.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this disclosure belongs. Although methods and materials similar or equivalent to
those described herein can be used in the practice of the disclosed methods and compositions, the exemplary methods, devices and materials are described herein.

The publications discussed above and throughout the text are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the inventors are not entitled to
antedate such disclosure by virtue of prior disclosure.

Subsequent studies of the S. aureus pigment have unraveled an elaborate biosynthetic pathway that produces a series of carotenoids. Similar carotenoids produced in dietary fruits and vegetables are well recognized as potent antioxidants by
virtue of their free radical scavenging properties and exceptional ability to quench singlet oxygen. The invention demonstrates that these anti-oxidant carotenoids produced by bacterial pathogens assist the pathogen in protecting itself from oxidative
burst during infection. Developing new classes of antibiotics that target bacterial virulence factors such as the S. aureus carotenoid pigment is an approach that has not been utilized.

Methods and compositions useful for treatment of microbial infections are provided. In one aspect the invention provides compositions and methods useful for treating a microbial infection wherein the microbe produces a carotenoid. For example,
methods and compositions useful for the treatment of S. aureus infections, including those produced by methicillin- and vancomycin-resistant strains, are provided by the invention. The methods and compositions of the invention can be used alone or in
combination with traditional antimicrobials and antibiotics to treat such infections. In addition, the methods and compositions disclosed herein can be used in settings such as foreign-body, catheter or endovascular infections, chronic osteomyeletis,
hospital acquired or post-operative infections, recurrent skin infections, or for S. aureus infections in the immunocompromised host. The invention demonstrates that by reducing the production of carotenoids there is a reduced resistance in microbial
that normal comprise such carotenoids to resist oxidative bursts.

A leading human pathogen Staphylococcus aureus (S. aureus) was named secondary to it characteristic golden yellow pigmentation (aureus=golden, Latin) in comparison to less virulent staphylococci (e.g. S. epidermidis) that normally colonize the
skin surface. Subsequent research regarding the S. aureus pigment unraveled an elaborate biosynthetic pathway that produces a series of carotenoids. Similar carotenoids produced in dietary fruits and vegetables are well recognized as potent
antioxidants by virtue of their free radical scavenging properties and exceptional ability to quench singlet oxygen. The S. aureus pigment has similar properties. The invention examined whether S. aureus could utilize its golden carotenoid pigment to
resist oxidant based clearance mechanisms of the host innate immune system. For example, neutrophils and macrophages kill bacteria by generating an "oxidative burst" of reactive molecules such as peroxide, bleach and singlet oxygen that kill bacteria
that they have phagocytosed.

Golden color imparted by carotenoid pigments is the eponymous feature of the human pathogen Staphylococcus aureus. A molecular genetic analysis pairing mutagenesis and heterologous expression was used to show that this hallmark phenotype is in
fact a virulence factor, serving to protect the bacterium from phagocytic killing through its antioxidant properties. In the present era, effective control of this important disease agent is compromised by rapid evolution of antimicrobial resistance in
both community and hospital settings. The invention demonstrates that the inhibition of carotenogenesis offers a therapeutic approach to the treatment of complicated S. aureus infections, effectively rendering the pathogen more susceptible to clearance
by normal host innate immune defenses.

Carotenoids represent a major class of natural pigments. More than 600 different carotenoids have been identified in bacteria, fungi, algae, plants and animals (Staub, O., In: Pfander, H. (ed.), Key to Carotenoids, 2.sup.nd ed., Birkhuser
Verlag, Basel). They function as accessory pigments in photosynthesis, as antioxidants, as precursors for vitamins in humans and animals and as pigments for light protection and species specific coloration. Carotenoids have historically been of
interest, e.g., for pharmaceuticals, food colorants, and animal feed and nutrient supplements. The discovery that these natural products can play an important role in the prevention of cancer and chronic disease (mainly due to their antioxidant
properties) and, more recently, that they exhibit significant tumor suppression activity due to specific interactions with cancer cells, has boosted interest in their pharmaceutical potential (Bertram, J. S., Nutr. Rev., 1999;57:182-191; Singh, et al.,
Oncology, 1998;12:1643-168; Rock, C. L., Pharmacol. Ther., 1997;75:185-197; Edge, et al., J. Photochem. Photobiol., 1997;41:189-200).

Carotenoid can be produced in recombinant microorganisms by combining biosynthetic genes from different organisms to create biosynthetic pathways. At present more than 150 genes for 24 carotenogenic enzymes (crt) have been isolated from
bacteria, plants, algae and fungi that can be used to engineer a variety of diverse carotenoids. For example, Staphylococcus aureus contains a bright orange pigment, staphyloxanthin. This pigment is thought to protect bacteria from attack by reactive
oxygen species of the host immune system. The pigment is produced by a series of enzymes including dehydrosqualene synthase, which condenses two molecules of farnesyldiphosphate to form dehydrosqualene. These pigments are not produced by humans and
other animals since the dehydrosqualene synthase gene is absent in animals. However, humans and other animals do contain a squalene synthase gene. Remarkably, the invention demonstrates that inhibitors of the human squalene synthase enzyme block
pigment (staphyloxanthin) formation in S. aureus, leading to enhanced killing by immune system cells, both in vitro and in vivo.

The invention demonstrates that carotenoid pigment production contributes to resistance of Group B Streptococcus (GBS) to macrophage killing. GBS is the leading cause of invasive bacterial infections such as pneumonia, sepsis and meningitis in
human neonates. A gene required for production of carotenoid pigment (CylE) has been identified. The pigment is required for the organism's production of a hemolysin/cytolysin toxin that have cell damaging and proinflammatory properties important in
disease pathogenesis. Other bacterial and fungal human pathogens associated with invasive infections produce carotenoid or melanin-like pigments with antioxidant properties (e.g. Aspergillus fumigatus, Burkholderia cepacia, Serratia marcesens),
suggesting a common pathogenic theme and revealing a potential selective advantage for pigment production against phagocytic clearance mechanisms.

Accordingly, the approach of targeting pigment production for antimicrobial therapy can be extended to organisms that produce pigments (e.g., carotenoid pigments or other pigments that confer oxidative protection). In particular, Aspergillus
sp. are an important cause of mortality in immunocompromised subjects (e.g., cancer chemotherapy) and Burkholderia cepacia is an important cause of mortality in cystic fibrosis. Both Aspergillus and Burkholderia are commonly multidrug resistant and
recalcitrant to existing antibiotic therapies.

Using a molecular genetic approach of targeted gene deletion (to create nonpigmented S. aureus mutants) and cloning techniques (to express the S. aureus pigment in other bacteria), the invention provides a "living reagents" useful for screening
and identifying the importance of the cartenoid pigments (e.g., pigment in S. aureus disease pathogenesis). For example, using the methods of the invention S. aureus carotenoid pigment was demonstrated to protect the bacterium against peroxide and
singlet oxygen and made it more resistant to killing in mouse and human blood and by purified human neutrophils. Using a mouse model of S. aureus abscess formation, it was demonstrated that the carotenoid pigment promoted bacterial survival and disease
progression in vivo. Control experiments using mice impaired in neutrophil oxidant production showed that carotenoid pigment production contributed to S. aureus virulence by virtue of its antioxidant properties.

The invention demonstrates that pharmacological inhibition of S. aureus pigment production rendered the bacterium more susceptible to oxidants and impaired bacterial survival in blood. The invention further demonstrates that squalene synthesis
inhibitors can block Staphylococcus aureus pigment production, and that squalene synthesis inhibition can reduce Staphylococcus aureus pathogenicity in vivo.

One important mechanism by which phagocytic cells eliminate pathogens is through release of reactive oxygen species generated by NADPH oxidase. The invention demonstrates that carotenoids such as those expressed by S. aureus serve a protective
function against these defense molecules. For example, a .DELTA.CrtM mutant was killed 10 to 100-fold more efficiently by hydrogen peroxide and 100 to 1,000-fold more efficiently by singlet oxygen than the WT S. aureus strain. Similarly, heterologous
expression of staphylococcal pigment in S. pyogenes was associated with a 100 to 1,000-fold decrease in singlet oxygen lethality.

The invention provides methods and agents that inhibited carotenogenesis for the treatment of microbial infections and to facilitate anti-microbial activity. For example, in one aspect, the mixed function oxidase inhibitor
2-diethylaminoethyl-2,2-diphenyl-valerate (SKF 525-A, Calbiochem) is shown to inhibit pigment formation in S. aureus, and a dose-dependent decrease in carotenoid production was demonstrated in the WT strain of S. aureus grown in the presence of the
agent. Blocking S. aureus pigment formation led to a commensurate dose-dependent increase in the susceptibility of the organism to singlet oxygen killing and a decrease in its ability to survive in human whole blood. As a control, the .DELTA.CrtM
mutant was exposed to SKF 525-A in parallel experiments with no significant effects on oxidant susceptibility or blood survival.

The invention provides methods and compositions useful for treating bacterial infection by inhibiting the production and/or activity of carotenoids. More particularly, the invention provides methods of treating a subject having a bacterial
infection (e.g., a bacterial infection of Staphylococcus sp.) comprising contacting a subject with an agent that inhibits the activity and/or production of a carotenoid of the Staphylococcus sp. In one aspect, the agent is a small molecule, a
polynucleotide (e.g., a ribozyme or antisense molecule), a polypeptide (e.g., an antibody) or a peptidomimetic. In one aspect of the invention, the agent is a pharmacologic agent that inhibits carotenogenesis. For example, the agent can be a mixed
function oxidase inhibitor such as 2-diethylaminoethyl-2,2-diphenyl-valerate (SKF 525-A, Calbiochem), 2,4-dichloro-6-phenylphenoxyethylamine, 2,4-dichloro-6-phenylphenoxyethyldiethylamine, and piperonyl butoxide. In yet another aspect, the agent can
comprise a squalene synthetase inhibitor. The squalene synthetase inhibitors suitable for use herein include, but are not limited to, .alpha.-phosphono-sulfonates disclosed in U.S. Pat. No. 5,712,396, those disclosed by Biller et al, J. Med. Chem.,
1988, Vol. 31, No. 10, pp 1869-1871, including isoprenoid (phosphinyl-methyl)phosphonates, as well as other known squalene synthetase inhibitors, for example, as disclosed in U.S. Pat. Nos. 4,871,721 and 4,924,024 and in Biller, S. A., Neuenschwander,
K., Ponpipom, M. M., and Poulter, C. D., Current Pharmaceutical Design, 2, 1-40 (1996). In addition, other squalene synthetase inhibitors suitable for use herein include the terpenoid pyrophosphates disclosed by P. Ortiz de Montellano et al, J. Med.
Chem., 1977, 20, 243-249, the farnesyl diphosphate analog A and presqualene pyrophosphate (PSQ-PP) analogs as disclosed by Corey and Volante, J. Am. Chem. Soc., 1976, 98, 1291-1293, phosphinylphosphonates reported by McClard, R. W. et al, J.A.C.S.,
1987, 109, 5544 and cyclopropanes reported by Capson, T. L., PhD dissertation, June, 1987, Dept. Med. Chem. U of Utah, Abstract, Table of Contents, pp 16, 17, 40-43, 48-51, Summary.

Small molecule crtM inhibitors include the following and derivatives and salts thereof. A crtM inhibitor of the disclosure has a general formula:

The invention provides compounds and methods of using such compounds to treat microbial infections. The compounds of the invention are represented by formula I or II:

##STR00005## wherein: m is 0, 1, 2 or 3; n is an integer between 1 and 10 inclusive; each D and E are each independently selected from the group consisting of: H, aryl, substituted aryl, alkyl, substituted alkyl, carboxyl, aminocarbonyl,
alkylsulfonylaminocarboxyl, alkoxycarbonyl, and halo; M.sup.1, M.sup.2, and M.sup.3 are each independently selected from the group consisting of metals, ammonium and esters thereof; R.sup.1 is selected from the group consisting of: H, aryl, substituted
aryl, alkyl, substituted alkyl, carboxyl, aminocarbonyl, alkylsulfonylaminocarboxyl, alkoxycarbonyl, and halo, or R.sup.1 and R.sup.2, together with the carbons to which they are bound, can be joined to form a 4 to 7 membered ring or a substituted 4 to 7
membered ring; R.sup.2 is selected from the group consisting of: H, aryl, substituted aryl, alkyl, substituted alkyl, carboxyl, aminocarbonyl, alkylsulfonylaminocarboxyl, alkoxycarbonyl, and halo, or R.sup.2 and R.sup.1, together with the carbons to
which they are bound, can be joined to form a 4 to 7 membered ring or a substituted 4 to 7 membered ring, or R.sup.2 and R.sup.3, together with the carbons to which they are bound, can be joined to form a 4 to 7 membered ring or a substituted 4 to 7
membered ring; R.sup.3 is selected from the group consisting of: H, aryl, substituted aryl, alkyl, substituted alkyl, carboxyl, aminocarbonyl, alkylsulfonylaminocarboxyl, alkoxycarbonyl, and halo, or R.sup.3 and R.sup.2, together with the carbons to
which they are bound, can be joined to form a 4 to 7 membered ring or a substituted 4 to 7 membered ring, or R.sup.3 and R.sup.4, together with the carbons to which they are bound, can be joined to form a 4 to 7 membered ring or a substituted 4 to 7
membered ring; R.sup.4 is selected from the group consisting of: H, aryl, substituted aryl, alkyl, substituted alkyl, carboxyl, aminocarbonyl, alkylsulfonylaminocarboxyl, alkoxycarbonyl, and halo, or R.sup.4 and R.sup.3, together with the carbons to
which they are bound, can be joined to form a 4 to 7 membered ring or a substituted 4 to 7 membered ring; R.sup.5, R.sup.6, R.sup.7, R.sup.8, and R.sup.9 are each independently selected from the group consisting of: H, aryl, substituted aryl, alkyl,
substituted alkyl, carboxyl, aminocarbonyl, alkylsulfonylaminocarboxyl, alkoxycarbonyl, and halo; L.sup.1 is --S--, --SO--, --SO.sub.2--, --O--, --N(R.sup.19)--, or --C(R.sup.20)(R.sup.21)--; wherein R.sup.19, R.sup.20 and R.sup.21 are each independently
selected from the group consisting of: H, aryl, substituted aryl, alkyl, substituted alkyl, carboxyl, aminocarbonyl, alkylsulfonylaminocarboxyl, alkoxycarbonyl, and halo;

##STR00006## wherein: x is 0, 1, 2, or 3; y is an integer between 1 and 10 inclusive; each G and J are independently selected from the group consisting of: H, aryl, substituted aryl, alkyl, substituted alkyl, carboxyl, aminocarbonyl,
alkylsulfonylaminocarboxyl, alkoxycarbonyl, and halo; M.sup.4, M.sup.5, and M.sup.6 are each independently selected from the group consisting of metals, ammonium and esters thereof; R.sup.10 is selected from the group consisting of: H, aryl, substituted
aryl, alkyl, substituted alkyl, carboxyl, aminocarbonyl, alkylsulfonylaminocarboxyl, alkoxycarbonyl, and halo, or R.sup.10 and R.sup.11, together with the carbons to which they are bound, can be joined to form a 4 to 7 membered ring or a substituted 4 to
7 membered ring; R.sup.11 is selected from the group consisting of: H, aryl, substituted aryl, alkyl, substituted alkyl, carboxyl, aminocarbonyl, alkylsulfonylaminocarboxyl, alkoxycarbonyl, and halo, or R.sup.11 and R.sup.10, together with the carbons to
which they are bound, can be joined to form a 4 to 7 membered ring or a substituted 4 to 7 membered ring, or R.sup.11 and R.sup.12, together with the carbons to which they are bound, can be joined to form a 4 to 7 membered ring or a substituted 4 to 7
membered ring; R.sup.12 is selected from the group consisting of: H, aryl, substituted aryl, alkyl, substituted alkyl, carboxyl, aminocarbonyl, alkylsulfonylaminocarboxyl, alkoxycarbonyl, and halo, or R.sup.12 and R.sup.11, together with the carbons to
which they are bound, can be joined to form a 4 to 7 membered ring or a substituted 4 to 7 membered ring, or R.sup.12 and R.sup.13, together with the carbons to which they are bound, can be joined to form a 4 to 7 membered ring or a substituted 4 to 7
membered ring; R.sup.13 is selected from the group consisting of: H, aryl, substituted aryl, alkyl, substituted alkyl, carboxyl, aminocarbonyl, alkylsulfonylaminocarboxyl, alkoxycarbonyl, and halo, or R.sup.13 and R.sup.12, together with the carbons to
which they are bound, can be joined to form a 4 to 7 membered ring or a substituted 4 to 7 membered ring; R.sup.14, R.sup.15, R.sup.16, R.sup.17, and R.sup.18 are each independently selected from the group consisting of: H, aryl, substituted aryl, alkyl,
substituted alkyl, carboxyl, aminocarbonyl, alkylsulfonylaminocarboxyl, alkoxycarbonyl, and halo; L.sup.2 is --S--, --SO--, --SO.sub.2--, --O--, --N(R.sup.22)--, or --C(R.sup.23)(R.sup.24)--; wherein R.sup.22, R.sup.23 and R.sup.24 are each independently
selected from the group consisting of: H, aryl, substituted aryl, alkyl, substituted alkyl, carboxyl, aminocarbonyl, alkylsulfonylaminocarboxyl, alkoxycarbonyl, and halo.

In another aspect, of the invention, m is zero or one; n is and integer between 1 and 5, inclusive; each D and E are independently selected from H, alkyl, substituted alkyl and halo; R.sup.1, R.sup.2, R.sup.3, R.sup.4, R.sup.5, R.sup.6, R.sup.7,
R.sup.8, and R.sup.9 are each independently selected from the group consisting of: H, aryl, substituted aryl, alkyl, substituted alkyl, and halo; R.sup.19, R.sup.20 and R.sup.21 are each independently selected from the group consisting of: H, alkyl, and
substituted alkyl; M.sup.1, M.sup.2, and M.sup.3 are each independently alkali metals; x is zero or one; y is and integer between 1 and 5, inclusive; each G and J are independently selected from H, alkyl, substituted alkyl and halo; R.sup.10, R.sup.11,
R.sup.12, R.sup.13, R.sup.14, R.sup.15, R.sup.16, R.sup.17, and R.sup.18 are each independently selected from the group consisting of: H, aryl, substituted aryl, alkyl, substituted alkyl, and halo; R.sup.22, R.sup.23 and R.sup.24 are each independently
selected from the group consisting of: H, alkyl, and substituted alkyl; M.sup.4, M.sup.5, and M.sup.6 are each independently alkali metals.

In yet another aspect, m is one; n is three; each D and E are H; R.sup.1 is H, F, or benzyl; R.sup.2 is H, F or CF.sub.3; R.sup.3 is H, F, CL, CF.sub.3, t-butyl, n-propyl, or benzyl; R.sup.4 is H or F; R.sup.5, R.sup.6, R.sup.7 and R.sup.9 are
each H; R.sup.8 is H or F; M.sup.1, M.sup.2, and M.sup.3 are each potassium; L.sup.1 is --O--, --NH-- or CH.sub.2; x is zero or one; y is three; each G and J are H; R.sup.10, R.sup.11, R.sup.13, R.sup.14, R.sup.15, R.sup.16, R.sup.17, and R.sup.18 are
each H; R.sup.12 is H or CH.sub.3 M.sup.4, M.sup.5, and M.sup.6 are each potassium; L.sup.2 is --O--.

Alkyl groups include straight-chain, branched and cyclic alkyl groups. Alkyl groups include those having from 1 to 20 carbon atoms. Alkyl groups include small alkyl groups having 1 to 3 carbon atoms. Alkyl groups include medium length alkyl
groups having from 4-10 carbon atoms. Alkyl groups include long alkyl groups having more than 10 carbon atoms, particularly those having 10-20 carbon atoms. Cyclic alkyl groups include those having one or more rings. Cyclic alkyl groups include those
having a 3-, 4-, 5-, 6-, 7-, 8-, 9- or 10-member carbon ring and particularly those having a 3-, 4-, 5-, 6-, or 7-member ring. The carbon rings in cyclic alkyl groups can also carry alkyl groups. Cyclic alkyl groups can include bicyclic and tricyclic
alkyl groups. Alkyl groups optionally include substituted alkyl groups. Substituted alkyl groups include among others those which are substituted with aryl groups, which in turn can be optionally substituted. Specific alkyl groups include methyl,
ethyl, n-propyl, iso-propyl, cyclopropyl, n-butyl, s-butyl, t-butyl, cyclobutyl, n-pentyl, branched-pentyl, cyclopentyl, n-hexyl, branched hexyl, and cyclohexyl groups, all of which are optionally substituted. The term cyclopentyl ring refers to a ring
of five carbons with any degree of unsaturation. The term cyclohexyl ring refers to a ring of six carbons with any degree of unsaturation.

Alkenyl groups include straight-chain, branched and cyclic alkenyl groups. Alkenyl groups include those having 1, 2 or more double bonds and those in which two or more of the double bonds are conjugated double bonds. Alkenyl groups include
those having from 2 to 20 carbon atoms. Alkenyl groups include small alkyl groups having 2 to 3 carbon atoms. Alkenyl groups include medium length alkenyl groups having from 4-10 carbon atoms. Alkenyl groups include long alkenyl groups having more
than 10 carbon atoms, particularly those having 10-20 carbon atoms. Cyclic alkenyl groups include those having one or more rings. Cyclic alkenyl groups include those in which a double bond is in the ring or in an alkenyl group attached to a ring.
Cyclic alkenyl groups include those having a 3-, 4-, 5-, 6-, 7-, 8-, 9- or 10-member carbon ring and particularly those having a 3-, 4-, 5-, 6- or 7-member ring. The carbon rings in cyclic alkenyl groups can also carry alkyl groups. Cyclic alkenyl
groups can include bicyclic and tricyclic alkyl groups. Alkenyl groups are optionally substituted. Substituted alkenyl groups include among others those which are substituted with alkyl or aryl groups, which groups in turn can be optionally
substituted. Specific alkenyl groups include ethenyl, prop-1-enyl, prop-2-enyl, cycloprop-1-enyl, but-1-enyl, but-2-enyl, cyclobut-1-enyl, cyclobut-2-enyl, pent-1-enyl, pent-2-enyl, branched pentenyl, cyclopent-1-enyl, hex-1-enyl, branched hexenyl,
cyclohexenyl, all of which are optionally substituted.

Aryl groups include groups having one or more 5- or 6-member aromatic or heteroaromatic rings. Aryl groups can contain one or more fused aromatic rings. Heteroaromatic rings can include one or more N, O, or S atoms in the ring. Heteroaromatic
rings can include those with one, two or three N, those with one or two O, and those with one or two S. Aryl groups are optionally substituted. Substituted aryl groups include among others those which are substituted with alkyl or alkenyl groups, which
groups in turn can be optionally substituted. Specific aryl groups include phenyl groups, biphenyl groups, pyridinyl groups, and naphthyl groups, all of which are optionally substituted.

Alkylaryl groups are aryl groups substituted with one or more alkyl groups wherein the alkyl groups optionally carry additional substituents and the aryl groups are optionally substituted. Specific alkylaryl groups are alkyl-substituted phenyl
groups such as methylphenyl.

The rings that may be formed from two or more of R1-R5 together can be optionally substituted cycloalkyl groups, optionally substituted cycloalkenyl groups or aromatic groups. The rings may contain 3, 4, 5, 6, 7 or more carbons. The rings may
be heteroaromatic in which one, two or three carbons in the aromatic ring are replaced with N, O or S. The rings may be heteroalkyl or heteroalkenyl, in which one or more CH2 groups in the ring are replaced with O, N, NH, or S.

Optional substitution of any alkyl, alkenyl and aryl groups includes substitution with one or more of the following substituents: halogens, --CN, --COOR, --OR, --COR, --OCOOR, --CON(R)2, --OCON(R)2, --N(R)2, --NO2, --SR, --SO2R, --SO2N(R)2 or
--SOR groups. Optional substitution of alkyl groups includes substitution with one or more alkenyl groups, aryl groups or both, wherein the alkenyl groups or aryl groups are optionally substituted. Optional substitution of alkenyl groups includes
substitution with one or more alkyl groups, aryl groups, or both, wherein the alkyl groups or aryl groups are optionally substituted. Optional substitution of aryl groups includes substitution of the aryl ring with one or more alkyl groups, alkenyl
groups, or both, wherein the alkyl groups or alkenyl groups are optionally substituted.

Optional substituents for alkyl, alkenyl and aryl groups include among others: --COOR where R is a hydrogen or an alkyl group or an aryl group and more specifically where R is methyl, ethyl, propyl, butyl, or phenyl groups all of which are
optionally substituted; --COR where R is a hydrogen, or an alkyl group or an aryl groups and more specifically where R is methyl, ethyl, propyl, butyl, or phenyl groups all of which groups are optionally substituted; --CON(R).sub.2 where each R,
independently of each other R, is a hydrogen or an alkyl group or an aryl group and more specifically where R is methyl, ethyl, propyl, butyl, or phenyl groups all of which groups are optionally substituted; R and R can form a ring which may contain one
or more double bonds; --OCON(R).sub.2 where each R, independently of each other R, is a hydrogen or an alkyl group or an aryl group and more specifically where R is methyl, ethyl, propyl, butyl, or phenyl groups all of which groups are optionally
substituted; R and R can form a ring which may contain one or more double bonds; --N(R).sub.2 where each R, independently of each other R, is a hydrogen, or an alkyl group, acyl group or an aryl group and more specifically where R is methyl, ethyl,
propyl, butyl, or phenyl or acetyl groups all of which are optionally substituted; or R and R can form a ring which may contain one or more double bonds. --SR, --SO.sub.2R, or --SOR where R is an alkyl group or an aryl groups and more specifically where
R is methyl, ethyl, propyl, butyl, phenyl groups all of which are optionally substituted; for --SR, R can be hydrogen; --OCOOR where R is an alkyl group or an aryl groups; --SO.sub.2N(R).sub.2 where R is a hydrogen, an alkyl group, or an aryl group and R
and R can form a ring; --OR where R.dbd.H, alkyl, aryl, or acyl; for example, R can be an acyl yielding --OCOR* where R* is a hydrogen or an alkyl group or an aryl group and more specifically where R* is methyl, ethyl, propyl, butyl, or phenyl groups all
of which groups are optionally substituted.

Compounds of the invention can have prodrug forms. Prodrugs of the compounds of the invention are useful in the methods of this invention. Any compound that will be converted in vivo to provide a biologically, pharmaceutically or
therapeutically active form of a compound of the invention is a prodrug. Various examples and forms of prodrugs are well known in the art. Examples of prodrugs are found, inter alia, in Design of Prodrugs, edited by H. Bundgaard, (Elsevier, 1985),
Methods in Enzymology, Vol. 42, at pp. 309-396, edited by K. Widder, et. al. (Academic Press, 1985); A Textbook of Drug Design and Development, edited by Krosgaard-Larsen and H. Bundgaard, Chapter 5, "Design and Application of Prodrugs," by H.
Bundgaard, at pp. 113-191, 1991); H. Bundgaard, Advanced Drug Delivery Reviews, Vol. 8, p. 1-38 (1992); H. Bundgaard, et al., Journal of Pharmaceutical Sciences, Vol. 77, p. 285 (1988); and Nogrady (1985) Medicinal Chemistry A Biochemical Approach,
Oxford University Press, New York, pages 388-392).

The invention also provides compounds comprising the following structures. Such compounds are useful inhibitors of dehydrosqualene synthase activity (crtM inhibitors). The methods and compositions of the invention include enantiomers,
pharmaceutically acceptable salts and derivatives thereof.

In addition, phosphonosulfinic acid squalene synthase inhibitors, such as those disclosed in U.S. Pat. No. 5,447,922, and the following Chemical Abstract Registry numbers, can be used in the methods of the invention: 172152-68-0P;
172152-69-1P; 172152-70-4P; 172152-71-5P; 172152-72-6P; 172152-73-7P; 172152-74-8P; 172152-75-9P; 172152-76-0P; 172152-77-1P; 172152-78-2P; 172152-79-3P; 172152-80-6P; 172152-81-7P; 172152-82-8P; 172152-83-9P; 172152-84-0P; 172152-85-1P; and
172152-86-2P.

Referring to FIG. 1, additional enzymatic steps can be inhibited in addition to crtM to render a microbe susceptible to oxidative damage. For example, inhibitors of crtN can be used alone or in combination with a crtM. For example,
diphenylamine and diphenylamine derivatives and analogs such as diclofenac sodium, mefenamic acid and lobenzarit disodium can be used to inhibit staphyloxanthin production. As yet another example, bisphosphonates are well known as inhibitors of farnesyl
diphosphate synthase the previous enzyme in the production of staphyloxanthin. The use of inhibitors in combination with crtM inhibitors can act synergistically with CrtM inhibitors to render a microbe that produces a carotenoid susceptible to oxidative
damage.

Compounds of the invention can have prodrug forms. Prodrugs of the compounds of the invention are useful in the methods of this invention. Any compound that will be converted in vivo to provide a biologically, pharmaceutically or
therapeutically active form of a compound of the invention is a prodrug. Various examples and forms of prodrugs are well known in the art. Examples of prodrugs are found, inter alia, in Design of Prodrugs, edited by H. Bundgaard, (Elsevier, 1985),
Methods in Enzymology, Vol. 42, at pp. 309-396, edited by K. Widder, et. al. (Academic Press, 1985); A Textbook of Drug Design and Development, edited by Krosgaard-Larsen and H. Bundgaard, Chapter 5, "Design and Application of Prodrugs," by H.
Bundgaard, at pp. 113-191, 1991); H. Bundgaard, Advanced Drug Delivery Reviews, Vol. 8, p. 1-38 (1992); H. Bundgaard, et al., Journal of Pharmaceutical Sciences, Vol. 77, p. 285 (1988); and Nogrady (1985) Medicinal Chemistry A Biochemical Approach,
Oxford University Press, New York, pages 388-392).

The invention also provides a method of preventing or treating a bacterial infection, comprising administering to a subject inflicted with the infection an agent that inhibits the production and/or activity of a carotenoid in the bacteria. In
one embodiment, the bacterial infection is a Staphylococcus infection. In another embodiment, the bacteria is a Staphylococcus sp. In yet a further embodiment, the bacteria is Staphylococcus aureus.

Within the context of the invention, modulating the expression of bacterial carotenoid biosynthetic pathways by the use of inhibitory nucleic acids provides a useful therapeutic method. For example, the invention identifies a number of genes
encoding enzymes in the carotenoid biosynthetic pathway including crtM and crtN. Alternatively (or in addition to), it may be desirable to knockout the crtM/crtN genes leading to the synthesis of C.sub.30 carotenoids. Common molecular biology
techniques can be used to generate inhibitory nucleic acid molecules such as, for example, antisense molecules that can interact with a crtN and/or M nucleic acid produced by wild type bacteria (e.g., Staphylococcus sp.).

In another aspect, the carotenogenesis inhibitor comprises a oligonucleotide or polynucleotide (e.g., an antisense, ribozyme, or siRNA). For example, antisense technology is a method of down-regulating genes where the sequence of the target
gene is known. A large number of carotenoid genes are known in the art including those of Staphylococcus sp. In this aspect, a nucleic acid segment from the desired gene (e.g., crtM and/or crtN) is cloned and operably linked to a promoter such that the
anti-sense strand of RNA will be transcribed. This construct is then introduced into a target cell and the antisense strand of RNA is produced. Antisense RNA inhibits gene expression by preventing the accumulation of mRNA encoding the protein of
interest. Thus, an antisense molecule against crtM and/or crtN will result in the reduction of synthesis of carotenoids a pathogenic microbe, thereby rendering the microbe susceptible to oxidative damage by phagocytes. A person skilled in the art will
know that certain considerations are associated with the use of antisense technologies in order to reduce expression of particular genes. For example, the proper level of expression of antisense genes may require the use of different chimeric genes
utilizing different regulatory elements known to the skilled artisan.

As used herein an isolated nucleic acid is substantially free of proteins, lipids, and other nucleic acids with which an in vivo-produced nucleic acids naturally associated. Typically, the nucleic acid is at least 70%, 80%, 90% or more pure by
weight, and conventional methods for synthesizing nucleic acids in vitro can be used in lieu of in vivo methods. As used herein, "nucleic acid" or "polynucleotide" or "oligonucleotide" refers to a polymer of deoxyribonucleotides or ribonucleotides, in
the form of a separate fragment or as a component of a larger genetic construct (e.g., by operably linking a promoter to a nucleic acid encoding, for example, an antisense molecule). Numerous genetic constructs (e.g., plasmids and other expression
vectors) are known in the art and can be used to produce a desired nucleic acid of the disclosure in cell-free systems or prokaryotic or eukaryotic (e.g., yeast, insect, or mammalian) cells. The nucleic acids of the disclosure can readily be used in
conventional molecular biology methods to produce the peptides of the disclosure.

Polynucleotides comprising an antisense nucleic acid, ribozyme, or siRNA can be inserted into an "expression vector." The term "expression vector" refers to a genetic construct such as a plasmid, virus or other vehicle known in the art that can
be engineered to contain, for example, an antisense molecule. The expression vector typically contains an origin of replication, and a promoter, as well as genes that allow phenotypic selection of the transformed cells. Various promoters, including
inducible and constitutive promoters, can be utilized in the disclosure. Typically, the expression vector contains a replicon site and control sequences that are derived from a species compatible with the host/target cell.

Transformation or transfection of a host/target cell with a polynucleotide of the disclosure can be carried out using conventional techniques known to those skilled in the art. For example, where the host cell is E. coli, competent cells that
are capable of DNA uptake can be prepared using the CaCl.sub.2, MgCl.sub.2 or RbCl methods known in the art. Alternatively, physical means, such as electroporation or microinjection can be used. Electroporation allows transfer of a polynucleotide into
a cell by high voltage electric impulse. Additionally, polynucleotides can be introduced into host cells by protoplast fusion, using methods well known in the art. Suitable methods for transforming eukaryotic cells, such as electroporation and
lipofection, also are known.

A host cell or target cell encompassed by of the disclosure are any cells in which the polynucleotides of the disclosure can be used to express an inhibitory nucleic acid molecule. The term also includes any progeny of a host/target cell.
Host/target cells, which are useful, include bacterial cells, fungal cells (e.g., yeast cells), plant cells and animal cells. For example, host cells can be a higher eukaryotic cell, such as a mammalian cell, or a lower eukaryotic cell, such as a yeast
cell, or the host cell can be a prokaryotic cell, such as a bacterial cell. Introduction of the construct into the host cell can be effected by calcium phosphate transfection, DEAE-Dextran mediated transfection, or electroporation (Davis, L., Dibner,
M., Battey, I., Basic Methods in Molecular Biology (1986)). As representative examples of appropriate hosts, there may be mentioned: fungal cells, such as yeast; insect cells such as Drosophila S2 and Spodoptera Sf9; animal cells such as CHO, COS or
Bowes melanoma; plant cells, and the like. The selection of an appropriate host is deemed to be within the scope of those skilled in the art from the teachings herein.

The activity of such inhibitory agents (e.g., small molecules (2-diethylaminoethyl-2,2-diphenyl-valerate and inhibitory nucleic acids) can be determined using conventional methods known to those of skill in the art, such as the assays described
herein (both in vitro and in vivo assays). For example, in one aspect of the invention, a method to screen an agent useful for treating an infection comprising contacting a microbe with an inhibitory agent the inhibits carotenoid production under
conditions wherein the inhibitory agent and microbe can interact, contacting the microbe with an agent the peroxide, singlet oxygen or oxidative bursts and measuring the microbes survival in the presence and absence of the inhibitory agents, wherein a
decrease in survival is indicative of an agent that promotes antimicrobial activity.

The disclosure also provides a method for inhibiting the growth of a bacterium by contacting the bacterium with an inhibiting effective amount of a carotenoid biosynthesis inhibitor (i.e., a carotenogenesis inhibitor). The term "contacting"
refers to exposing the microbe (e.g., bacterium) to an agent so that the agent can inhibit, kill, or lyse microbe or render it susceptible to oxidative destruction. Contacting of an organism with an agent that inhibits carotenoid biosynthesis can occur
in vitro, for example, by adding the agent to a bacterial culture, or contacting a bacterially contaminated surface with the agent.

Alternatively, contacting can occur in vivo, for example, by administering the agent to a subject afflicted with a bacterial infection or susceptible to infection. In vivo contacting includes both parenteral as well as topical. "Inhibiting" or
"inhibiting effective amount" refers to the amount of agent that is sufficient to cause, for example, a bacteriostatic or bactericidal effect, reduce coloration of a particular bacterial cell type, or decrease the amount of a particular carotenoid
produced by the bacteria. Bacteria that can be affected by the use of a carotenoid inhibitor include both gram-negative and gram-positive bacteria. For example, bacteria that can be affected include Staphylococcus aureus, Streptococcus pyogenes (group
A), Streptococcus sp. (viridans group), Streptococcus agalactiae (group B), S. bovis, Streptococcus (anaerobic species), Streptococcus pneumoniae, and Enterococcus sp.; Gram-negative cocci such as, for example, Neisseria gonorrhoeae, Neisseria
meningitidis, and Branhamella catarrhalis; Gram-positive bacilli such as Bacillus anthracis, Bacillus subtilis, P. acne Corynebacterium diphtheriae and Corynebacterium species which are diptheroids (aerobic and anerobic), Listeria monocytogenes,
Clostridium tetani, Clostridium difficile, Escherichia coli, Enterobacter species, Proteus mirablis and other sp., Pseudomonas aeruginosa, Klebsiella pneumoniae, Salmonella, Shigella, Serratia, and Campylobacter jejuni. In particular, the methods and
compositions of the invention are useful against any pathogen that synthesizes a carotenoid the confers protection against reactive oxygen species (e.g., species produced by NADPH oxidase). Infection with one or more of these bacteria can result in
diseases such as bacteremia, pneumonia, meningitis, osteomyelitis, endocarditis, sinusitis, arthritis, urinary tract infections, tetanus, gangrene, colitis, acute gastroenteritis, impetigo, acne, acne posacue, wound infections, born infections, fascitis,
bronchitis, and a variety of abscesses, nosocomial infections, and opportunistic infections. The method for inhibiting the growth of bacteria can also include contacting the bacterium with the peptide in combination with one or more antibiotics.

A carotenoid biosynthesis inhibitor (e.g., crtM inhibitor, crtN inhibitor and combinations thereof) can be administered to any host, including a human or non-human animal, in an amount effective to inhibit the production of carotenoids that
confer, for example, resistance to oxidative attack. In one aspect, the administration results in the inhibition of growth of a bacterium, virus, and/or fungus. Thus, the methods and compositions are useful as antimicrobial agents, antiviral agents,
and/or antifungal agents.

Any of a variety of art-known methods can be used to administer a carotenoid inhibitory agent either alone or in combination with other antibiotic agents. For example, administration can be parenterally by injection or by gradual infusion over
time. The agent(s) can be administered intravenously, intraperitoneally, intramuscularly, subcutaneously, intracavity, by inhalation, or transdermally.

In another aspect, a carotenoid biosynthesis inhibitor can be formulated either alone or in combination with other antibiotics/antifungals for topical administration (e.g., as a lotion, cream, spray, gel, or ointment). Such topical formulations
are useful in treating or inhibiting microbial, fungal, and/or viral presence or infections on the eye, skin, and mucous membranes (e.g., mouth, vagina). Examples of formulations in the market place include topical lotions, creams, soaps, wipes, and the
like. It may be formulated into liposomes to reduce toxicity or increase bioavailability. Other methods for delivery include oral methods that entail encapsulation of the in microspheres or proteinoids, aerosol delivery (e.g., to the lungs), or
transdermal delivery (e.g., by iontophoresis or transdermal electroporation). Other methods of administration will be known to those skilled in the art.

Preparations for parenteral administration of a composition comprising a carotenoid inhibitor include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene
glycol, vegetable oils (e.g., olive oil), and injectable organic esters such as ethyl oleate. Examples of aqueous carriers include water, saline, and buffered media, alcoholic/aqueous solutions, and emulsions or suspensions. Examples of parenteral
vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, and fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's
dextrose), and the like. Preservatives and other additives such as, other antimicrobial, anti-oxidants, cheating agents, inert gases and the like also can be included.

The disclosure provides a method for inhibiting a bacterial, viral and/or fungal-associated disorder by contacting or administering a therapeutically effective amount of a carotenoid biosynthesis inhibitor (e.g., a crtM and/or crtN inhibitor)
either alone or in combination with other antimicrobial agents to a subject who has, or is at risk of having, such a disorder. The term "inhibiting" means preventing or ameliorating a sign or symptoms of a disorder (e.g., a rash, sore, and the like).
Examples of disease signs that can be ameliorated include an increase in a subject's blood level of TNF, fever, hypotension, neutropenia, leukopenia, thrombocytopenia, disseminated intravascular coagulation, adult respiratory distress syndrome, shock,
and organ failure. Examples of subjects who can be treated in the disclosure include those at risk for, or those suffering from, a toxemia, such as endotoxemia resulting from a gram-negative or gram-positive bacterial infection. Other examples include
subjects having a dermatitis as well as those having skin infections or injuries subject to infection with gram-positive or gram-negative bacteria, a virus, or a fungus. Examples of candidate subjects include those suffering from infection by E. coli,
Neisseria meningitides, staphylococci, or pneumococci. Other subjects include those suffering from gunshot wounds, renal or hepatic failure, trauma, burns, immunocompromising infections (e.g., HIV infections), hematopoietic neoplasias, multiple myeloma,
Castleman's disease or cardiac myxoma. Those skilled in the art of medicine can readily employ conventional criteria to identify appropriate subjects for treatment in accordance with the disclosure.

A therapeutically effective amount can be measured as the amount sufficient to decrease a subject's symptoms (e.g., dermatitis or rash by measuring the frequency of severity of skin sores). Typically, the subject is treated with an amount of a
therapeutic composition of the invention sufficient to reduce a symptom of a disease or disorder by at least 50%, 90% or 100%. Generally, the optimal dosage will depend upon the disorder and factors such as the weight of the subject, the type of
bacteria, virus or fungal infection, the weight, sex, and degree of symptoms. Nonetheless, suitable dosages can readily be determined by one skilled in the art. Typically, a suitable dosage is 0.5 to 40 mg/kg body weight, e.g., 1 to 8 mg/kg body
weight.

As mentioned previously, the compositions and methods of the invention can include the use of additional (e.g., in addition to a carotenoid biosynthesis inhibitor) therapeutic agents (e.g., an inhibitor of TNF, an antibiotic, and the like). The
carotenoid biosynthesis inhibitor, other therapeutic agent(s), and/or antibiotic(s) can be administered, simultaneously, but may also be administered sequentially. Suitable antibiotics include aminoglycosides (e.g., gentamicin), beta-lactams (e.g.,
penicillins and cephalosporins), quinolones (e.g., ciprofloxacin), and novobiocin. Generally, the antibiotic is administered in a bactericidal, antiviral and/or antifungal amount. Their effects can also be augmented by co-administration with an
inhibitor of flavohemoglobin, (Helmick et al., Imidazole antibiotics inhibit the nitric oxide dioxygenase function of microbial flavohemoglobin. Antimicrob Agents Chemother, 2005,49(5):1837-43, and Sud et al., Action of antifungal imidazoles on
Staphylococcus aureus, Antimicrob Agents Chemother, 1982, 22(3):470-4), increasing the efficacy of NO-based S. aureus killing by macrophages, and optionally triple combination therapies comprising one squalene synthase inhibitor, one flavohemoglobin
(nitric oxide dioxygenase) inhibitor such as an azole (miconazole, econazole, clortrimazole, and ketoconazole) and one antibiotic as described above, may be applied to a patient in need of therapy.

The methods and compositions of the invention utilizing carotenoid biosynthesis inhibitors are useful as a broad-spectrum antimicrobials suitable for tackling the growing problem of antibiotic-resistant bacteria strains, and for treating and/or
preventing outbreaks of infectious diseases, including diseases caused by bioterrorism agents like anthrax, plague, cholera, gastroenteritis, multidrug-resistant tuberculosis (MDR TB).

A pharmaceutical composition comprising a carotenogenesis inhibitor according to the disclosure can be in a form suitable for administration to a subject using carriers, excipients, and additives or auxiliaries. Frequently used carriers or
auxiliaries include magnesium carbonate, titanium dioxide, lactose, mannitol and other sugars, talc, milk protein, gelatin, starch, vitamins, cellulose and its derivatives, animal and vegetable oils, polyethylene glycols and solvents, such as sterile
water, alcohols, glycerol, and polyhydric alcohols. Intravenous vehicles include fluid and nutrient replenishers. Preservatives include antimicrobial, chelating agents, and inert gases. Other pharmaceutically acceptable carriers include aqueous
solutions, non-toxic excipients, including salts, preservatives, buffers and the like, as described, for instance, in Remington's Pharmaceutical Sciences, 15th ed., Easton: Mack Publishing Co., 1405-1412, 1461-1487 (1975), and The National Formulary
XIV., 14th ed., Washington: American Pharmaceutical Association (1975), the contents of which are hereby incorporated by reference. The pH and exact concentration of the various components of the pharmaceutical composition are adjusted according to
routine skills in the art. See Goodman and Gilman's, The Pharmacological Basis for Therapeutics (7th ed.).

The pharmaceutical compositions according to the disclosure may be administered locally or systemically. A "therapeutically effective dose" is the quantity of an agent according to the disclosure necessary to prevent, to cure, or at least
partially arrest the symptoms of a bacterial infection. Amounts effective for this use will, of course, depend on the severity of the disease and the weight and general state of the subject. Typically, dosages used in vitro may provide useful guidance
in the amounts useful for in situ administration of the pharmaceutical composition, and animal models may be used to determine effective dosages for treatment of infections. Various considerations are described, e.g., in Langer, Science, 249: 1527,
(1990); Gilman et al. (eds.) (1990), each of which is herein incorporated by reference.

As used herein, "administering a therapeutically effective amount" is intended to include methods of giving or applying a pharmaceutical composition of the disclosure to a subject that allow the composition to perform its intended therapeutic
function. The therapeutically effective amounts will vary according to factors, such as the degree of infection in a subject, the age, sex, and weight of the individual. Dosage regima can be adjusted to provide the optimum therapeutic response. For
example, several divided doses can be administered daily or the dose can be proportionally reduced as indicated by the exigencies of the therapeutic situation.

The pharmaceutical composition can be administered in a convenient manner, such as by injection (subcutaneous, intravenous, etc.), oral administration, inhalation, transdermal application, or rectal administration. Depending on the route of
administration, the pharmaceutical composition can be coated with a material to protect the pharmaceutical composition from the action of enzymes, acids, and other natural conditions that may inactivate the pharmaceutical composition. The pharmaceutical
composition can also be administered parenterally or intraperitoneally. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof, and in oils. Under ordinary conditions of storage and use, these preparations may
contain a preservative to prevent the growth of microorganisms.

Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases,
the composition should be sterile and should be fluid to the extent that easy syringability exists. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid
polyetheylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size, in the case of
dispersion, and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases,
it will be typical to include isotonic agents, for example, sugars, polyalcohols, such as mannitol, sorbitol, or sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition
an agent that delays absorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the pharmaceutical composition in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered
sterilization. Generally, dispersions are prepared by incorporating the pharmaceutical composition into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above.

The pharmaceutical composition can be orally administered, for example, with an inert diluent or an assimilable edible carrier. The pharmaceutical composition and other ingredients can also be enclosed in a hard or soft-shell gelatin capsule,
compressed into tablets, or incorporated directly into the individual's diet. For oral therapeutic administration, the pharmaceutical composition can be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches,
capsules, elixirs, suspensions, syrups, wafers, and the like. Such compositions and preparations should contain at least 1% by weight of active compound. The percentage of the compositions and preparations can, of course, be varied and can conveniently
be between about 5% to about 80% of the weight of the unit.

The tablets, troches, pills, capsules, and the like can also contain the following: a binder, such as gum gragacanth, acacia, corn starch, or gelatin; excipients such as dicalcium phosphate; a disintegrating agent, such as corn starch, potato
starch, alginic acid, and the like; a lubricant, such as magnesium stearate; and a sweetening agent, such as sucrose, lactose or saccharin, or a flavoring agent such as peppermint, oil of wintergreen, or cherry flavoring. When the dosage unit form is a
capsule, it can contain, in addition to materials of the above type, a liquid carrier. Various other materials can be present as coatings or to otherwise modify the physical form of the dosage unit. For instance, tablets, pills, or capsules can be
coated with shellac, sugar, or both. A syrup or elixir can contain the agent, sucrose as a sweetening agent, methyl and propylparabens as preservatives, a dye, and flavoring, such as cherry or orange flavor. Of course, any material used in preparing
any dosage unit form should be pharmaceutically pure and substantially non-toxic/biocompatible in the amounts employed. In addition, the pharmaceutical composition can be incorporated into sustained-release preparations and formulations.

Thus, a "pharmaceutically acceptable carrier" is intended to include solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. The use of such media and agents for
pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the pharmaceutical composition, use thereof in the therapeutic compositions and methods of treatment is contemplated.
Supplementary active compounds can also be incorporated into the compositions.

It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. "Dosage unit form" as used herein, refers to physically discrete units suited as unitary dosages for the
individual to be treated; each unit containing a predetermined quantity of pharmaceutical composition is calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit
forms of the disclosure are related to the characteristics of the pharmaceutical composition and the particular therapeutic effect to be achieve.

The principal pharmaceutical composition is compounded for convenient and effective administration in effective amounts with a suitable pharmaceutically acceptable carrier in an acceptable dosage unit. In the case of compositions containing
supplementary active ingredients, the dosages are determined by reference to the usual dose and manner of administration of the said ingredients.

An agent useful to inhibit carotenogenesis in a bacterial organism (e.g., Staphylococcus sp.) may be used in combination with commonly used antibiotics and/or antimicrobials. Accordingly, a pharmaceutical composition of the invention can
comprise a carotenogenesis inhibitor and one or more additional antimicrobials or antibiotics.

The following example(s) are provided in illustration of the invention and should not be construed in any way as constituting a limitation thereof.

EXAMPLES

The biosynthetic pathway for S. aureus carotenoids includes the essential functions of genes crtM and crtN, encoding dehydrosqualene synthase and dehydrosqualene desaturase, respectively. To probe the biological activities of the S. aureus
pigment, an isogenic mutant of a golden-colored human clinical isolate by allelic replacement of crtM was generated. The .DELTA.CrtM mutant was nonpigmented and lacked the characteristic triple-peak spectral profile of wild-type carotenoid at 440, 462
and 491 nM wavelengths. No differences in growth rate, stationary phase density, surface charge, buoyancy or hydrophobicity were observed between WT and .DELTA.CrtM S. aureus. S. aureus crtM and crtN together are sufficient for production of
4,4'-diaponeurosporene. To facilitate gain of function analyses, both genes were expressed in the nonpigmented Streptococcus pyogenes, a human pathogen associated with a disease spectrum similar to that of S. aureus. When transformed with a pCrtMN
plasmid, S. pyogenes gained yellow pigmentation with the spectral characteristics of a carotenoid. Complementation of the S. aureus .DELTA.CrtM mutant with the same pCrtMN vector also partially restored pigmentation.

The isoprene biosynthesis pathway is an important target for the development of drugs effective against infectious diseases, (U.S. Patent application 20030032578), as well as well as in the development of herbicides which interfere with orange
pigment (carotenoid) formation (U.S. Pat. No. 5,756,423).

Useful inhibitors of carotenogenesis production include various small molecule inhibitors. For example, a small molecule of formula I, provides a generic formula of a molecule useful for inhibiting carotenogenesis production.

The invention provides compounds and methods of using such compounds to treat microbial infections. The compounds of the invention are represented by formula I or II:

##STR00010## wherein: m is 0, 1, 2 or 3; n is an integer between 1 and 10 inclusive; each D and E are each independently selected from the group consisting of: H, aryl, substituted aryl, alkyl, substituted alkyl, carboxyl, aminocarbonyl,
alkylsulfonylaminocarboxyl, alkoxycarbonyl, and halo; M.sup.1, M.sup.2, and M.sup.3 are each independently selected from the group consisting of metals, ammonium and esters thereof; R.sup.1 is selected from the group consisting of: H, aryl, substituted
aryl, alkyl, substituted alkyl, carboxyl, aminocarbonyl, alkylsulfonylaminocarboxyl, alkoxycarbonyl, and halo, or R.sup.1 and R.sup.2, together with the carbons to which they are bound, can be joined to form a 4 to 7 membered ring or a substituted 4 to 7
membered ring; R.sup.2 is selected from the group consisting of: H, aryl, substituted aryl, alkyl, substituted alkyl, carboxyl, aminocarbonyl, alkylsulfonylaminocarboxyl, alkoxycarbonyl, and halo, or R.sup.2 and R.sup.1, together with the carbons to
which they are bound, can be joined to form a 4 to 7 membered ring or a substituted 4 to 7 membered ring, or R.sup.2 and R.sup.3, together with the carbons to which they are bound, can be joined to form a 4 to 7 membered ring or a substituted 4 to 7
membered ring; R.sup.3 is selected from the group consisting of: H, aryl, substituted aryl, alkyl, substituted alkyl, carboxyl, aminocarbonyl, alkylsulfonylaminocarboxyl, alkoxycarbonyl, and halo, or R.sup.3 and R.sup.2, together with the carbons to
which they are bound, can be joined to form a 4 to 7 membered ring or a substituted 4 to 7 membered ring, or R.sup.3 and R.sup.4, together with the carbons to which they are bound, can be joined to form a 4 to 7 membered ring or a substituted 4 to 7
membered ring; R.sup.4 is selected from the group consisting of: H, aryl, substituted aryl, alkyl, substituted alkyl, carboxyl, aminocarbonyl, alkylsulfonylaminocarboxyl, alkoxycarbonyl, and halo, or R.sup.4 and R.sup.3, together with the carbons to
which they are bound, can be joined to form a 4 to 7 membered ring or a substituted 4 to 7 membered ring; R.sup.5, R.sup.6, R.sup.7, R.sup.8, and R.sup.9 are each independently selected from the group consisting of: H, aryl, substituted aryl, alkyl,
substituted alkyl, carboxyl, aminocarbonyl, alkylsulfonylaminocarboxyl, alkoxycarbonyl, and halo; L.sup.1 is --S--, --SO--, --SO.sub.2--, --O--, --N(R.sup.19)--, or --C(R.sup.20)(R.sup.21)--; wherein R.sup.19, R.sup.20 and R.sup.21 are each independently
selected from the group consisting of: H, aryl, substituted aryl, alkyl, substituted alkyl, carboxyl, aminocarbonyl, alkylsulfonylaminocarboxyl, alkoxycarbonyl, and halo;

##STR00011## wherein: x is 0, 1, 2, or 3; y is an integer between 1 and 10 inclusive; each G and J are independently selected from the group consisting of: H, aryl, substituted aryl, alkyl, substituted alkyl, carboxyl, aminocarbonyl,
alkylsulfonylaminocarboxyl, alkoxycarbonyl, and halo; M.sup.4, M.sup.5, and M.sup.6 are each independently selected from the group consisting of metals, ammonium and esters thereof; R.sup.10 is selected from the group consisting of: H, aryl, substituted
aryl, alkyl, substituted alkyl, carboxyl, aminocarbonyl, alkylsulfonylaminocarboxyl, alkoxycarbonyl, and halo, or R.sup.10 and R.sup.11, together with the carbons to which they are bound, can be joined to form a 4 to 7 membered ring or a substituted 4 to
7 membered ring; R.sup.11 is selected from the group consisting of: H, aryl, substituted aryl, alkyl, substituted alkyl, carboxyl, aminocarbonyl, alkylsulfonylaminocarboxyl, alkoxycarbonyl, and halo, or R.sup.11 and R.sup.10, together with the carbons to
which they are bound, can be joined to form a 4 to 7 membered ring or a substituted 4 to 7 membered ring, or R.sup.11 and R.sup.12, together with the carbons to which they are bound, can be joined to form a 4 to 7 membered ring or a substituted 4 to 7
membered ring; R.sup.12 is selected from the group consisting of: H, aryl, substituted aryl, alkyl, substituted alkyl, carboxyl, aminocarbonyl, alkylsulfonylaminocarboxyl, alkoxycarbonyl, and halo, or R.sup.12 and R.sup.11, together with the carbons to
which they are bound, can be joined to form a 4 to 7 membered ring or a substituted 4 to 7 membered ring, or R.sup.12 and R.sup.13, together with the carbons to which they are bound, can be joined to form a 4 to 7 membered ring or a substituted 4 to 7
membered ring; R.sup.13 is selected from the group consisting of: H, aryl, substituted aryl, alkyl, substituted alkyl, carboxyl, aminocarbonyl, alkylsulfonylaminocarboxyl, alkoxycarbonyl, and halo, or R.sup.13 and R.sup.12, together with the carbons to
which they are bound, can be joined to form a 4 to 7 membered ring or a substituted 4 to 7 membered ring; R.sup.14, R.sup.15, R.sup.16, R.sup.17, and R.sup.18 are each independently selected from the group consisting of: H, aryl, substituted aryl, alkyl,
substituted alkyl, carboxyl, aminocarbonyl, alkylsulfonylaminocarboxyl, alkoxycarbonyl, and halo; L.sup.2 is --S--, --SO--, --SO.sub.2, --O--, --N(R.sup.22)--, or --C(R.sup.23)(R.sup.24)--; wherein R.sup.22 R.sup.23 and R.sup.24 are each independently
selected from the group consisting of: H, aryl, substituted aryl, alkyl, substituted alkyl, carboxyl, aminocarbonyl, alkylsulfonylaminocarboxyl, alkoxycarbonyl, and halo. Structures of CrtM Inhibitors and Their Inhibitory Activity ( M)

##STR00012## ##STR00013## ##STR00014##

Many infections are caused by bacteria containing pigments. For example, Staphylococcus aureus contains a bright orange pigment, staphyloxanthin. This pigment is thought to protect bacteria from attack by reactive oxygen species of the host
immune system. The pigment is produced by a series of enzymes including dehydrosqualene synthase, which condenses two molecules of farnesyldiphosphate to form dehydrosqualene. These pigments are not produced by humans and other animals since the
dehydrosqualene synthase gene is absent in animals. However, humans and other animals do contain a squalene synthase gene. Remarkably, it has been determined that inhibitors of the human squalene synthase enzyme block pigment (staphyloxanthin)
formation in S. aureus, leading to enhanced killing by immune system cells, both in vitro and in vivo.

The compound of Formula III was prepared as described in Magnin et al., .alpha.-Phosphonosulfonic acids: Potent and selective inhibitors of squalene synthase, J. Med. Chem. 39 (1996) 657-660, and tested in S. aureus growth inhibition and in
pigment formation. The compound of Formula III had no detectable effect on cell growth but had a potent effect on pigment formation, Table 1/Figure 1:

Human CGD patient: The patient was an 18 y.o. female with a gp47.sup.phox deficiency (homozygous .DELTA.GT deletion in exon 2). At the time of study she was in good health and her only medication was interferon-.gamma. (50 mcg/m.sup.2)
administered three times weekly by subcutaneous injection.

Generation of the carotenoid-deficient S. aureus mutant, .DELTA.CrtM. Precise, in-frame allelic replacement of the S. aureus crtM gene with a chloramphenicol acetyltransferase (cat) cassette was performed using PCR-based methods as described
for S. pyogenes or Streptococcus agalactiae, with minor modifications. Primers were designed based on the published S. aureus crtMN sequence.sup.6 cross-referenced to genome S. aureus strain N315.sup.21. PCR was used to amplify .about.500 bp upstream
of crtM with primers crtMupF 5'-TTAGGAAGTGCATATACTTCAC-3' (SEQ ID NO:1) and crtMstartR 5'-GGTGGTATATCCAGTGATTTTTTTCTCCATACTAGTCCTCCTATATTGAAATG-3' (SEQ ID NO:2), along with approximately 500 bp of sequence immediately downstream of crtM with primers
crtMendF 5'-TACTGCGATGAGTGGCAGGGCGGGGCGTAACAAAGTATTTAGTATTGAAGC-3' (SEQ ID NO:3) and crtMdownR 5'-GGCACCGTTATACGATCATCGT-3' (SEQ ID NO:4). The crtMstartR and crtMendF primers were constructed with 25 bp 5' extensions corresponding to the 5' and 3' ends
of the cat gene, respectively. The upstream and downstream PCR products were then combined with a 650 bp amplicon of the complete cat gene (from pACYC184) as templates in a second round of PCR using primers crtMupF and crtMdownR. The resultant PCR
amplicon, containing an in-frame substitution of crtM with cat, was subcloned into temperature-sensitive vector pHY304 to create the knockout plasmid. This vector was transformed initially into permissive S. aureus strain RN4220 (provided by Dr. Paul
Sullam) and then into S. aureus strain Pig1 by electroporation. Transformants were grown at 30.degree. C., shifted to the nonpermissive temperature for plasmid replication (40.degree. C.), and differential antibiotic selection and pigment phenotype
were used to identify candidate mutants. Allelic replacement of the crtM allele in was confirmed unambigously by PCR reactions documenting targeted insertion of cat and absence of crtM in chromosomal DNA isolated from the final mutant .DELTA.CrtM.

Complementation and heterologous expression studies. Primers CrtF 5'-CAGTCTAGAAATGGCATTTCAATATAGGAG-3' (SEQ ID NO:5) and CrtR 5'-ATCGAGATCTCTCACATCTTTCTCTTAGAC-3' (SEQ ID NO:6) were used to amplify the contiguous CrtM and CrtN genes from the
chromosome of WT S. aureus strain Pig1. The fragment was directionally cloned into the shuttle expression vector pDCerm.sup.19 and the recombinant plasmid (pCrtMN) used to transform by electroporation the S. aureus .DELTA.CrtM mutant and S. pyogenes
strain 5448.

Spectral profile of the S. aureus carotenoid. Stationary phase (48 h) cultures of WT S. aureus Pig1 and its isogenic .DELTA.CrtM mutant were subjected to methanol extraction. The absorbance profile of the extracts was measured with a MBA 2000
spectrophotometer (Perkin Elmer).

Oxidant susceptibility assays. Tests for susceptibility to oxidants were performed either in PBS (S. aureus) or THB (S. pyogenes). Hydrogen peroxide (H.sub.2O.sub.2) was added to 1.5% final concentration, 2.times.0.sup.9 bacteria incubated at
37.degree. C. for 1 h, then 1,000 U/ml of catalase (Sigma) added to quench residual H.sub.2O.sub.2. Dilutions were plated on THA for enumeration of surviving cfu. For the singlet oxygen assay, 10.sup.8 S. aureus or 4.times.10.sup.8 S. pyogenes were
incubated at 37.degree. C. in individual wells of a 24-well culture plate in the presence or absence of 1-6 .mu.g/ml methylene blue and situated exactly 10 cm from a 100-watt light source. Bacterial viability was assessed after 1-3 h by plating
dilutions on THA. Control plates handled identically but wrapped in foil or exposed to light in the absence of methylene blue did not show evidence of bacterial killing.

Whole blood killing assays. Bacteria were washed twice in PBS, diluted to an inoculum of 10.sup.4 cfu in 25 .mu.l PBS, and mixed with 75 .mu.l of freshly drawn human or mouse blood in heparinized tubes. The tubes were incubated at 37.degree.
C. for 4 h with agitation, at which time dilutions were plated on THA for enumeration of surviving cfu.

Neutrophil intracellular survival assay. Neutrophils were purified from healthy human volunteers using a Histopaque gradient (Sigma) per manufacturer's directions. Intracellular survival assays were performed as follows. Bacterial cultures
were washed twice in PBS, diluted to a concentration of 4.5.times.10.sup.6 cfu in 100 .mu.l RPMI+10% FCS, and mixed with 3.times.10.sup.5 neutrophils in the same media (multiplicity of infection, MOI=15:1), centrifuged at 700.times.g for 5 min, then
incubated at 37.degree. C. in a 5% CO.sub.2 incubator. Gentamicin (Gibco) (final concentration 400 .mu.g/ml for S. aureus and 100 .mu.g/ml for S. pyogenes) was added after 10 min to kill extracellular bacteria. At specified time points, the contents
of sample wells were withdrawn, centrifuged to pellet the neutrophils, and washed to remove the antibiotic medium. Neutrophils were then lysed in 0.02% triton-X, and cfu calculated by plating on THA. Several assays were repeated with addition of a step
involving preincubation of the bacterial inoculum with 10% autologous human serum for 15 min on ice.

Murine model of subcutaneous infection. Ten to 16 week old CD-1 or gp91.sup.Phox-/- mice were injected subcutaneously in one flank (chosen randomly) with the bacterial test strain, and simultaneously in the opposite flank with a different
strain for direct comparison. Bacterial cultures were washed, diluted and resuspended in PBS mixed 1:1 with sterile Cytodex beads (Amersham) at the specified inoculum, following an established protocol for generating localized S. aureus and S. pyogenes
subcutaneous infection. Lesion size, as assessed by the maximal length.times.width of the developing ulcers, was recorded daily. Cumulative lesion size represents the total sum of lesion sizes from all animals in each treatment group on a given day.
At day 8 (S. aureus) or day 5 (S. pyogenes), animals were euthanized, skin lesions excised, homogenized in PBS, and plated on THA for quantitative culture.

Statistics. The significance of experimental differences in oxidant sensitivity, blood killing, and neutrophil survival were evaluated by unpaired Students t test. Results of the mouse in vivo challenge studies were evaluated by paired
Student's t test.

Assurances. All animal experiments were approved by the UCSD Committee on the Use and Care of Animals and performed using accepted veterinary standards. Experimentations using human blood were approved by the Dual Tracked UCSD Human Research
Protection Program/CHSD IRB. Prior informed consents were obtained from the human subjects.

Assays for buoyancy, surface charge, and hydrophobicity. To measure buoyancy, sequential overlay gradients of 1 ml each 70%, 60% and 50% Percoll were prepared in 5 ml glass test tubes. One ml of overnight bacterial culture was placed on top of
the Percoll layers, the tubes centrifuged in a swinging bucket centrifuge for 8 min at 500.times.g, and the migration of bacteria to various Percoll interphases recorded. To measure surface charge, bacteria were harvested by centrifugation and washed in
morpholinepropanesulfonic acid (MOPS) buffer (20 mM, pH=7.0). One ml of culture was resuspended in 0.5 ml MOPS and the OD600 measured. Bacteria cells were incubated at room temperature or 15 min with cytochrome C (Sigma, St. Louis, Mo.) at a final
concentration of 0.5 mg/ml. Samples were centrifuged (13,000.times.g for 5 min) and the amount of cytochrome C remaining in the supernatant quantitated at 530 nm. Cytochrome C values were adjusted to reflect binding per culture OD.sub.600=1.0. To
measure hydrophobicity, 0.5 ml of S. aureus culture was washed and resuspended in 1.0 ml PBS, 300 .mu.l of n-hexadecane layered on top of the cell suspension, and tubes vortexed for 60 sec. Samples were incubated at RT for 30 min to allow for phase
separation. The aqueous phase was removed and the ratio of the OD.sub.600 of the aqueous phase versus the OD.sub.600 of the culture in PBS determined.

Protease and cathelicidin sensitivity assays. Human neutrophil elastase, and cathepsin G were purchased from Calbiochem. The antimicrobial peptide mCRAMP was synthesized at the Louisiana State University Protein Facility (Martha Juban,
Director). S. aureus cultures were diluted (1:2,000) in 10 mM phosphate buffer (pH 7.2)+0.5% LB to .about.1.times.10.sup.6 CFU/ml. Ninety .mu.l of this bacterial suspension was added to replicate wells in a 96-well plate. Dilutions of the cathepsin G
(20 and 100 mU/ml), human neutrophil elastase (12.5 and 50 .mu.g/ml), and murine CRAMP (0.4-3 .mu.M) were prepared in 10 mM phosphate buffer and added to wells in 10 .mu.l volume; 10 mM phosphate buffer alone was used as a negative control. After 2 h
incubation at 37.degree. C., 25 .mu.l aliquots of each well were serially diluted in PBS and plated on THB. Each experiment was performed in duplicate and repeated.

Phagocytotic uptake assay. S. aureus were labeled with SYTOR9, a component of BacLight.TM. kit (Invitrogen, Carlsbad, Calif.), for 15 min at RT per manufacturer's guidelines. Labeled bacteria were washed.times.3 times to remove excess dye,
then preopsonized in 10% autologous human serum for 10 min on ice. Bacteria were added to 3.times.106 purified human neutrophils at MOI=15, incubated at 37.degree. C. for 5 min, then centrifuged at 500.times.g for 6 min to pellet neutrophils. The
supernatant was discarded and the cell pellet resuspended in 15 ml of 0.1 mg/ml ethidium bromide in PBS to quench fluorescence of extracellular bacteria. The percentage of neutrophils with intracellular bacteria was enumerated by direct visualization
under fluorescent microscopy. Experiments were performed in duplicate and repeated. Representative images were captured on using Delta Vision Deconvolution Microscope System (Nikon TE-200 Microscope) at the UCSD Digital Imaging Core Facility.

The biosynthetic pathway for S. aureus carotenoids.sup.6 includes the essential functions of genes crtM and crtN, encoding dehydrosqualene synthase and dehydrosqualene desaturase, respectively (FIG. 1a). To probe the biological activities of
the S. aureus pigment, an isogenic mutant of a golden-colored human clinical isolate by allelic replacement of crtM was generated (FIG. 1a). Consistent with previous reports, pigmentation of the wild-type (WT) strain became apparent in early stationary
phase of growth and continued to intensify before reaching a plateau at 36-48 h (FIG. 1b). The .DELTA.CrtM mutant was nonpigmented and lacked the characteristic triple-peak spectral profile of wild-type carotenoid at 440, 462 and 491 nM wavelengths
(FIG. 1b). No differences in growth rate, stationary phase density, surface charge, buoyancy or hydrophobicity were observed between WT and .DELTA.CrtM S. aureus (FIGS. 5a-d). S. aureus crtM and crtN together are sufficient for production of
4,4'-diaponeurosporene. To facilitate gain of function analyses, both genes were expressed in the nonpigmented Streptococcus pyogenes, a human pathogen associated with a disease spectrum similar to that of S. aureus. When transformed with the pCrtMN
plasmid, S. pyogenes gained yellow pigmentation (FIG. 1b) with the spectral characteristics of a carotenoid. Complementation of the S. aureus .DELTA.CrtM mutant with pCrtMN vector also fully restored pigmentation (FIG. 1b).

One important mechanism by which phagocytic cells eliminate pathogens is through release of reactive oxygen species generated by NADPH oxidase. It has been suggested that bacterial carotenoids such as those expressed by S. aureus could serve a
protective function against these defense molecules. To test this experimentally, the susceptibility of WT and .DELTA.CrtM S. aureus to oxidants in vitro was compared. As shown in FIGS. 1c and 1d, the .DELTA.CrtM mutant was killed more efficiently by
hydrogen peroxide and singlet oxygen compared to the WT S. aureus strain. Complementation with pCrtMN restored the ability of the .DELTA.CrtM mutant to resist singlet oxygen killing (FIG. 1d). Similarly, heterologous expression of staphylococcal
pigment in S. pyogenes led to a significant decrease in susceptibility to singlet oxygen (FIG. 1e).

It was then determined whether the observed antioxidant activity of the S. aureus carotenoid translated to increased bacterial resistance to innate immune clearance using two ex vivo assay systems: human or mouse whole blood survival and
coculture with purified human neutrophils. WT S. aureus survived significantly better than the nonpigmented .DELTA.CrtM intracellularly within human neutrophils (FIG. 2a, FIG. 6f) and in whole blood of normal mice or human donors (FIGS. 2b, e). The
former effect was not explained by differences in the rate of phagocytosis, since uptake of the WT S. aureus and .DELTA.CrtM mutant was comparable (FIG. 6a). Nor were differences attributable to changes in the magnitude of neutrophil oxidative burst,
since uptake of WT and mutant strains produced similar results in a nitroblue tetrazolium (NBT) reduction assay (FIG. 6b). Complementation of the S. aureus .DELTA.CrtM mutant with pCrtMN restored resistance to killing by mouse whole blood (FIG. 2b).
Likewise, the pigmented S. pyogenes expressing staphylococcal carotenoid showed enhanced survival in human neutrophils versus the parent strain (FIG. 2c).

To verify the association of S. aureus carotenoid expression with enhanced phagocyte resistance was a direct consequence of its antioxidant properties, assays were repeated in the presence of the oxidative burst inhibitor diphenyleneiodonium
(DPI). WT and .DELTA.CrtM S. aureus survived equally well in human neutrophils (FIG. 2d) and mouse blood (FIG. 6d) when oxidative burst was inhibited by DPI. Gp47.sup.Phox-/- is an inherited defect in phagocyte oxidative burst function commonly found
in chronic granulomatous disease (CGD) patients, and the gp91.sup.Phox-/- mouse represents a model of human X-linked CGD. The survival advantage of WT over nonpigmented .DELTA.CrtM S. aureus was evident only in the blood of normal humans and mice (CD1
or C57Bl/6), and not in the blood of a human gp47.sup.phox-/- patient or gp91.sup.Phox-/- mice lacking NADPH oxidase activity (FIG. 2e, f).

It was recently reported that the apparent neutrophil killing of pathogens by reactive oxygen species could largely reflect the activation of granule proteases mediated through changes in potassium flux. There was no difference in the
susceptibility of WT and .DELTA.CrtM S. aureus to the antimicrobial action of cathepsin G, and both strains were resistant to human neutrophil elastase as previously observed for S. aureus (FIG. 6d). Other effector molecules of mammalian neutrophils
critical to innate immune defense are the cathelicidin family of antimicrobial peptides. The carotenoid-deficient S. aureus mutant was equally susceptible to killing by the murine cathelicidin mCRAMP when compared to the WT strain (FIG. 6e). These
results support a primary role for the free-radical scavenging antioxidant properties of the S. aureus carotenoid in resistance to neutrophil-mediated killing.

The in vitro and ex vivo results demonstrate that S. aureus carotenoid is both necessary and sufficient to promote oxidant resistance and phagocyte survival. To assess the significance of these observations to disease pathogenesis, a murine
subcutaneous challenge model was developed. In these studies, individual animals were injected simultaneously in one flank with the WT S. aureus strain and the opposite flank with the .DELTA.CrtM mutant. At the site of WT injection (10.sup.6 cfu), mice
developed sizable abscess lesions reaching a cumulative size of 80 mm.sup.2 by day 4; injection of an equivalent inoculum of the carotenoid-deficient mutant on the contralateral flank failed to produce visible lesions (FIG. 3a). Quantitative culture
from skin lesions at two different challenge doses (10.sup.6 cfu to 10.sup.7 cfu) consistently demonstrated significantly higher numbers of surviving WT S. aureus compared to the .DELTA.CrtM mutant in the individual mice (FIG. 3a). To corroborate that
an antioxidant effect is key to the mechanism of protection afforded by the S. aureus carotenoid in vivo, the subcutaneous infection experiment was repeated in gp91.sup.Phox-/- mice. In the absence of host NADPH oxidase function, WT and .DELTA.CrtM
mutant S. aureus produced lesions of similar cumulative size and no survival advantage was detected on quantitative abscess culture (FIG. 3b). It was then determined that S. aureus carotenoid was sufficient to enhance bacterial virulence by comparing
the course of infection produced by S. pyogenes expressing CrtMN to controls transformed with vector alone. In FIG. 3c, lesions generated by the carotenoid-expressing strain were significantly larger and contained greater numbers of surviving bacteria
than those produced by the WT strain. Raw data from the in vivo experiments is provided in Table 2.

Given the protective effect provided to the bacteria by the golden yellow pigments, a pharmacologic agent that inhibits carotenogenesis might render S. aureus more susceptible to immune clearance. The mixed function oxidase inhibitor
2-diethylaminoethyl-2,2-diphenyl-valerate (SKF 525-A, Calbiochem) was previously shown to inhibit pigment formation in S. aureus, though a moderate residual accumulation of .delta. carotenoid intermediate was noted in those experiments. Shown in FIG.
4a, a dose-dependent decrease in pigment production in the WT strain of S. aureus grown in the presence of this agent was obtained. Blocking S. aureus pigment formation led to a dose-dependent increase in the susceptibility of the organism to singlet
oxygen killing (FIG. 4b), and a decrease in its ability of WT S. aureus to survive in human whole blood (FIG. 4c). As a control, the .DELTA.CrtM mutant was exposed to SKF 525-A in parallel experiments with no significant effects on oxidant
susceptibility or blood survival (FIG. 4b, c).

Golden color imparted by carotenoid pigments is the eponymous feature of the human pathogen Staphylococcus aureus. A molecular genetic analysis pairing mutagenesis and heterologous expression was performed to show that this hallmark phenotype
is in fact a virulence factor, serving to protect the bacterium from phagocytic killing through its antioxidant properties. In the present era, effective control of this important disease agent is compromised by rapid evolution of antimicrobial
resistance in both community and hospital settings. In principle, the inhibition of carotenogenesis may offer a novel therapeutic approach to the treatment of complicated S. aureus infections, effectively rendering the pathogen more susceptible to
clearance by normal host innate immune defenses.

In addition, WT S. aureus survived significantly better than the nonpigmented .DELTA.CrtM in whole blood of human donors or normal mice and intracellularly within human neutrophils. The latter effect was not explained by differences in the rate
of phagocytosis, since uptake of the WT S. aureus and .DELTA.CrtM mutant was comparable. Nor were differences attributable to changes in the magnitude of neutrophil oxidative burst, since uptake of WT and mutant strains produced similar results in a
nitroblue tetrazolium (NBT) reduction assay. Complementation of the S. aureus .DELTA.CrtM mutant with pCrtMN restored resistance to killing by mouse whole blood or human neutrophils. Likewise, the pigmented S. pyogenes expressing staphylococcal
carotenoid showed enhanced survival in human neutrophils compared to the parent strain.

To verify the association of S. aureus carotenoid expression with enhanced phagocyte resistance was a direct consequence of its antioxidant properties, assays were repeated in the presence of the oxidative burst inhibitor diphenyleneiodonium
(DPI). WT and .DELTA.CrtM S. aureus survived equally well in human neutrophils and mouse blood when oxidative burst was inhibited by DPI. The gp91.sup.Phox-/- mouse represents a model of human X-linked chronic granulomatous disease, an inherited defect
in phagocyte oxidative burst function. The survival advantage of WT over nonpigmented .DELTA.CrtM S. aureus was evident only in the blood of normal mice (CD1 or C57Bl/6), and not in the blood of gp91.sup.Phox-/- mice lacking NADPH oxidase activity.

It was recently reported that the apparent neutrophil killing of pathogens by reactive oxygen species could largely reflect the activation of granule proteases mediated through changes in potassium flux. No difference was identified in the
susceptibility of WT and .DELTA.CrtM S. aureus to the antimicrobial action of cathepsin G, and both strains were resistant to human neutrophil elastase as previously observed for S. aureus. Other effector molecules of mammalian neutrophils critical to
innate immune defense are the cathelicidin family of antimicrobial peptides. The carotenoid-deficient S. aureus mutant was equally susceptible to killing by the murine cathelicidin mCRAMP when compared to the WT strain. These results support a primary
role for the free-radical scavenging antioxidant properties of the S. aureus carotenoid in resistance to neutrophil-mediated killing.

The in vitro and ex vivo results demonstrate that S. aureus carotenoid is both necessary and sufficient to promote oxidant resistance and phagocyte survival. To assess the significance of these observations to disease pathogenesis, a murine
model for subcutaneous challenge was developed. In these studies, individual animals were injected simultaneously in one flank with the WT S. aureus strain and the opposite flank with the .DELTA.CrtM mutant. At the site of WT injection mice developed
sizable subcutaneous abscesses, injection of an equivalent inoculum of the carotenoid-deficient mutant on the contralateral flank failed to produce visible abscesses. Quantitative culture from skin lesions consistently demonstrated significantly higher
numbers of surviving WT S. aureus compared to the .DELTA.CrtM mutant in the individual mice. To corroborate that an antioxidant effect is key to the mechanism of protection afforded by the S. aureus carotenoid in vivo, the subcutaneous infection
experiment was repeated in gp91.sup.Phox-/- mice. In the absence of host NADPH oxidase function, WT and .DELTA.CrtM mutant S. aureus produced lesions of similar cumulative size and no survival advantage was detected on quantitative abscess cultures.
Furthermore, while S. pyogenes infection was associated with development of necrotic ulcers rather than abscess formation, lesions generated by the carotenoid-expressing strain were significantly larger and contained greater numbers of surviving bacteria
than those produced by the WT strain.

Given the protective effect provided to the bacteria by the golden yellow pigments, a pharmacologic agent that inhibited carotenogenesis was tested to determine whether the agent might render S. aureus more susceptible to immune clearance. The
mixed function oxidase inhibitor 2-diethylaminoethyl-2,2-diphenyl-valerate (SKF 525-A, Calbiochem) was previously shown to inhibit pigment formation in S. aureus, and a dose-dependent decrease in carotenoid production was demonstrated in the WT strain of
S. aureus grown in the presence of the agent. Blocking S. aureus pigment formation led to a commensurate dose-dependent increase in the susceptibility of the organism to singlet oxygen killing and a decrease in its ability to survive in human whole
blood. As a control, the .DELTA.CrtM mutant was exposed to SKF 525-A in parallel experiments with no significant effects on oxidant susceptibility or blood survival.

Because the first committed steps in staphyloxanthin biosynthesis (FPP.fwdarw.presqualene diphosphate.fwdarw.dehydrosqualene, FIG. 1a) appear remarkably similar to the first steps (FPP.fwdarw.presqualene diphosphate.fwdarw.squalene) in
cholesterol and ergosterol biosynthesis (FIG. 1b), known inhibitors of squalene synthase developed in the context of cholesterol lowering therapy and as anti-parasitic agents (inhibiting ergosterol biosynthesis in Trypanosoma cruzi, the causative agent
of Chagas' disease) can also inhibit CrtM (i.e. dehydrosqualene synthase), thereby blocking pigment formation and invasive potential. A series of known SQS inhibitors were tested for inhibition of staphyloxanthin biosynthesis. One inhibitor, (rac
BMS-187745) showed particularly potent effects, having an IC.sub.50 of .about.1 .mu.M (FIG. 7). Since Formula III (and its analogs) have been advanced through Phase II human clinical trials as cholesterol-lowering agents, the agent is clearly of
potential interest as a new lead compound inhibiting staphyloxanthin virulence factor formation in S. aureus. To confirm whether formula III actually inhibits CrtM, the S. aureus enzyme was expressed in E. coli and the protein isolated. Recombinant
CrtM was found to be enzymatically active and potently inhibited by formula II with an IC.sub.50 of 600 nM, corresponding to a K.sub.i of .about.1.5 nM, strongly supporting CrtM as the target of this phosphonosulfonate drug in S. aureus.

Inhibition of staphyloxanthin biosynthesis by formula III could represent a purely virulence factor-based therapy for S. aureus infection. Incubation with up to 2 mM formula III did not affect either S. aureus growth characteristics or survival
through 48 hr in culture. However, after incubation with 100 .mu.M formula III, the resulting non-pigmented S. aureus were .about.15-fold more susceptible to killing by 1.5% hydrogen peroxide and .about.4-fold less able to survive in freshly isolated
human whole blood than were normally pigmented S. aureus treated with PBS control. As expected, formula III had no effect on the growth of three human cell lines (MCF-7, NCI-H460 and SF-268) since there only cholesterol biosynthesis is targeted, and
cholesterol is generally abundant in serum (or diet).

Given the in vitro abilities of formula III treatment to increase S. aureus susceptibility to oxidants and blood (neutrophil-mediated) clearance, in vivo testing was performed. Under intraperitoneal challenge one group of mice (n=14) was
treated with 1 0.5 mg b.i.d. (days -1, 0, 1 and 2), and a second group (n=13) with equivalent volume injections of PBS control. Upon sacrifice at 72 h, S. aureus bacterial counts in the kidneys of mice treated with 1 were significantly lower than those
of the control group (P<0.001), with cfus in 8/13 mice below the detection threshold, to be compared with 2/14 in the control group.

The results show that a drug developed for therapy of hypercholesterolemia via inhibition of human squalene synthase also neutralizes S. aureus dehydro-squalene synthase (CrtM), blocking production of staphyloxanthin, an important virulence
factor for the bacterium. This provides a novel pharmacological lead and a basis for rational drug design against this major human pathogen, which has become increasingly resistant to conventional antibiotics. The data also show utility of an
anti-infective without direct bactericidal properties that functions, instead, to render the pathogen susceptible to normal host innate immune clearance. Such anti-infectives exert less selective pressure for evolution of resistance, and promise
increased specificity of action, reducing undesired activities against the normal human micro-flora.

Although a number of embodiments and features have been described above, it will be understood by those skilled in the art that modifications and variations of the described embodiments and features may be made without departing from the
teachings of the disclosure or the scope of the invention as defined by the appended claims.